Articles having localized molecules disposed thereon and methods of producing same

ABSTRACT

Methods of producing substrates having selected active chemical regions by employing elements of the substrates in assisting the localization of active chemical groups in desired regions of the substrate. The methods may include optical, chemical and/or mechanical processes for the deposition, removal, activation and/or deactivation of chemical groups in selected regions of the substrate to provide selective active regions of the substrate.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of application U.S. Ser. No.14/318,324, filed Jun. 27, 2014, which is a continuation of applicationU.S. Ser. No. 11/731,748, filed Mar. 29, 2007, entitled “ARTICLES HAVINGLOCALIZED MOLECULES DISPOSED THEREON AND METHODS OF PRODUCING SAME” byDavid R. Rank et al., which is a continuation-in-part of applicationU.S. Ser. No. 11/394,352, filed Mar. 30, 2006, entitled “ARTICLES HAVINGLOCALIZED MOLECULES DISPOSED THEREON AND METHODS OF PRODUCING SAME” byDavid R. Rank et al. Each of these applications is incorporated hereinby reference in its entirety for all purposes.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under NHGRI Grant No.R01-HG003710-01 awarded by the National Human Genome Research Institute(NHGRI) of the National Institutes of Health (NIH). The government hascertain rights in the invention. The preceding statement is included inaccordance with 37 C.F.R. 401.14(0(4) because one or more inventionsdescribed herein were made or developed with government grant support.This statement should not be construed as necessarily covering allinventions described herein.

FIELD OF THE INVENTION

The present invention relates to methods of producing substrates havingselected active chemical regions by employing elements of the substratesin assisting the localization of active chemical groups in desiredregions of the substrate. Methods that include optical, chemical and/ormechanical processes for the deposition, removal, activation and/ordeactivation of chemical groups in selected regions of the substrate toprovide selective active regions of the substrate are described.

BACKGROUND OF THE INVENTION

There are a wide range of analytical operations that may benefit fromthe ability to analyze the reaction of individual molecules, relativelysmall numbers of molecules, or molecules at relatively lowconcentrations. A number of approaches have been described for providingthese sparsely populated reaction mixtures. For example, in the field ofnucleic acid sequence determination, a number of researchers haveproposed single molecule or low concentration approaches to obtainingsequence information in conjunction with the template dependentsynthesis of nucleic acids by the action of polymerase enzymes.

The various different approaches to these sequencing technologies offerdifferent methods of monitoring only one or a few synthesis reactions ata time. For example, in some cases, the reaction mixture is apportionedinto droplets that include low concentrations of reactants. In otherapplications, certain reagents are immobilized onto surfaces such thatthey may be monitored without interference from other reactioncomponents in solution. In still another approach, optical confinementtechniques are used to ascertain signal information only from arelatively small number of reactions, e.g., a single molecule, within anoptically confined area. Notwithstanding the availability of theabove-described techniques, there are instances where furtherselectivity of reaction components for analysis would be desirable. Thepresent invention meets these and a variety of needs.

BRIEF SUMMARY OF THE INVENTION

The present invention generally provides methods of providing functionalsurface modifications, e.g., active chemical groups, in selected regionsof substrates, as well as the resultant substrates produced thereby, andthe use of such substrates.

In a first aspect, the present invention provides methods of producing asubstrate having selectively active regions thereon. The methodscomprise providing a substrate having a plurality of nanostructuresdefined thereon, wherein each of the nanostructures limits an ability ofa first activation controlling agent to provide active chemical groupson selected portions of a surface of the substrate. The substrate isthen exposed to at least the first activation controlling agent toselectively provide active chemical groups on the selected portions of asurface of the substrate. Active chemical groups include, for example,chemical functional groups, active molecules (e.g., enzymes), andcoupling or binding groups.

In another, related aspect, the invention provides methods of preparingan analytical substrate having selected active regions thereon. Themethod provides a substrate having an optical analysis structuredisposed thereon, the optical analysis structure providing enhancedoptical access to selected regions of the substrate. Surface functionalgroups are then provided that are capable of being activated ordeactivated by a first electromagnetic radiation, which is directed atthe substrate whereby the optical analysis structure directs theelectromagnetic radiation to selectively activate or deactivate thesurface functional groups in the selected regions of the substrate toprovide selectively active regions of the substrate.

Another aspect of the invention features a method of providing a zeromode waveguides having an active chemical surface substantially at abottom portion of the waveguide. The method comprises providing a zeromode waveguide disposed in a substrate, providing functional chemicalgroups on surfaces of the zero mode waveguide, and exposing firstportions, but not second portions, of the surfaces of the zero modewaveguide to an activation controlling agent that selectively activatesor deactivates the functional chemical groups on the first portions toprovide an active chemical surface substantially at a bottom portion ofthe zero mode waveguide.

The invention also provides substrates and devices made using suchmethods, including zero mode waveguide arrays that comprise a pluralityof zero mode waveguide cores disposed in a cladding layer, each corehaving a bottom surface, and a chemically active surface substantiallyonly within the cores.

The invention further provides methods of providing a substrate havingselected active regions disposed thereon that comprise providing asubstrate having an optical enhancement structure defined thereon, theoptical enhancement structure being capable of directing electromagneticradiation to provide an enhanced electromagnetic field proximal to aselected region of the substrate surface sufficient to create a trappingforce proximal to the selected region. Electromagnetic radiation isdirected at the substrate to provide the enhanced electromagnetic fieldat the selected region sufficient to create a trapping force upon anactive molecule proximal to the selected region. The active molecule isthen coupled to the selected region.

The invention additionally provides methods of producing a substratehaving selected active regions disposed thereon that comprise providinga substrate having a surface comprised of chemical functional groups,and a plurality of discrete nanoscale reaction regions defined thereon,and patterning one or more of a chemical functional group or anactivation controlling agent onto the surface in selected regions toprovide active chemical functional regions substantially only in thediscrete nanoscale reaction regions.

Another aspect of the invention provides methods of identifying asequence of a nucleic acid molecules. The method comprises providing aplurality of nucleic acid polymerase/template/primer complexes withindiscrete observation regions on a substrate, and detecting sequentialaddition of nucleotides or nucleotide analogs in a template dependentmanner to identify a sequence of incorporation of the nucleotides ornucleotide analogs in the plurality of observation regions. In suchmethods, the substrate has been prepared to substantially reduce one ormore of polymerase activity, polymerase presence, template presence, andprimer presence in regions outside of the observation regions.

Relatedly, the invention provides a method of identifying a sequence ofa nucleic acid molecule, comprising providing a plurality of nucleicacid polymerase/template/primer complexes within discrete observationregions on a substrate surface, and detecting sequential addition ofnucleotides or nucleotide analogs in a template dependent manner toidentify a sequence of incorporation of the nucleotides or nucleotideanalogs in the plurality of observation regions. In such methods, anintra-observation region barrier is provided upon the substrate surfacebetween at least first and second discrete observation regions tosubstantially prevent intra-observation region diffusion of one or morereactants or products.

The invention also provides methods of preferentially localizing desiredmolecules within an optical confinement disposed upon a substrate. Themethods comprise depositing the desired molecules over the surface ofthe substrate, and selectively removing the desired molecules from thesurface of the substrate that is not within the optical confinement. Inone aspect, the substrate comprises an opaque (non-transparent) layerand a transparent layer, and the optical confinement comprises a zeromode waveguide disposed through the opaque layer to the transparentlayer.

Selectively removing the desired molecules from the surface of thesubstrate that is not within the optical confinement optionally involvescontacting the substrate with a deactivation component coupled to anexclusionary component that is at least partially excluded from enteringinto the optical confinement. The deactivation component removes themolecules it can access from the surface (e.g., from the upper surfaceand upper walls of the core of a zero mode waveguide); it is preventedfrom accessing the optical confinement, or the entirety of the opticalconfinement, by the exclusionary component.

In one class of embodiments, the deactivation component comprises anenzyme, for example, a protease (e.g., a nonspecific or a site-specificprotease), a nuclease, or a carbohydrase. The exclusionary component isoptionally a large particle such as a bead, a macromolecule, or a rigidor semi-rigid elongated polymer. In one class of embodiments, theexclusionary component comprises a double-stranded nucleic acidmolecule, e.g., a double-stranded DNA molecule coupled to a proteasedeactivation component.

The desired molecules that are selectively immobilized can beessentially any molecules, for example, active molecules such as enzymes(e.g., nucleic acid polymerases) or molecules comprising binding orcoupling moieties (e.g., biotin molecules) which can be employed in turnto immobilize other molecules.

Further provided are methods of localizing a molecule within an opticalconfinement disposed on a substrate that comprise providing a lightactivated coupling group on the surface of the substrate, includingwithin the optical confinement. Activating radiation is then directed atthe substrate, wherein the optical confinement permits activatingradiation to be incident only within the optical confinement. Themolecule is then coupled to the light activated coupling group.

Another aspect of the invention provides methods of selectivelyimmobilizing a molecule of interest on a substrate. The methods compriseproviding a substrate having a first surface component and a secondsurface component where the first and second surface component havedifferent surface characteristics, and selectively coupling the moleculeof interest to the first surface component, based upon a differencebetween the surface characteristics of the first surface component andthe surface characteristics of the second surface component.

In one class of embodiments, the differing surface characteristicscomprise surface charge or electrostatic interactions at the surface.For example, the first surface component can have or acquire a negativesurface charge and the second surface component a positive surfacecharge (or vice versa). As another example, the surface components canhave different surface chemisorption characteristics; for example, thesecond surface component can have strong chemical affinity for aparticular group (e.g., phosphonate or phosphate groups), while thefirst surface component does not have strong affinity for the group.

The methods can be employed to selectively immobilize molecules ofinterest, e.g., in a ZMW or other hybrid substrate. Thus, for example,the substrate optionally comprises a layer of the second surfacecomponent on a layer of the first surface component, with a zero modewaveguide disposed through the second surface component layer to thefirst surface component layer. In such embodiments (among others), thefirst surface component can comprise SiO₂ and/or the second surfacecomponent can comprise a metal or metal oxide (e.g., aluminum oraluminum oxide). In embodiments in which the hybrid substrate is incontact with a solution, the substrate can comprise a first surfacecomponent that is a material with a point of zero charge below the pH ofthe solution (e.g. SiO₂ at pH>2) and/or a second surface component thatcomprises a metal oxide with a point of zero charge above the pH of thesolution (e.g. aluminum oxide at pH<8; other metal oxides that arepositively charged at neutral pH include, but are not limited to,thallium oxide, iron oxide, yttrium oxide, zinc oxide, lanthanum oxide,and magnesium oxide).

In certain embodiments, the molecule of interest associatespreferentially to the first surface component. For example, positivelycharged polymerase molecules preferentially associate with negativelycharged silicate over positively charged metal surfaces. In otherembodiments, the substrate is contacted with a first composition thatselectively associates with the first surface component based upon thedifference between the surface characteristics of the first and secondsurface components. The first composition can serve a blocking function,or it can associate, directly or indirectly, with the molecule ofinterest. Thus, for example, in one class of embodiments, the firstcomposition comprises a first coupling group, and selectively couplingthe molecule of interest to the first surface component comprisescoupling the molecule of interest to the first coupling group (e.g., afunctional chemical group, a binding group such as biotin, or the like).

In one class of embodiments, the first composition comprises a silane,e.g., for selective association with a silicate first surface component.Exemplary silanes include, but are not limited to, biotin-PEG-silanes.In another class of embodiments, the first composition comprises aphospholipid. In yet another class of embodiments, the first compositioncomprises polylysine-PEG or polylysine-PEG-biotin.

The methods optionally include contacting the substrate with a secondcomposition that selectively associates with the second surfacecomponent based upon the difference between the surface characteristicsof the first and second surface components. Treatment with the secondcomposition is optionally performed before or after coupling of themolecule of interest to the first surface component, including before orafter treatment with any first composition. In some embodiments, thesecond composition comprises a second coupling group, typicallydifferent from any first coupling group. Exemplary second compositionsinclude, e.g., polyelectrolytes and polyelectrolyte-PEG copolymers. Themethod optionally includes depositing a polyelectrolyte multilayer onthe second surface component.

Additional exemplary second compositions include compounds comprisingone or more phosphonic acid or one or more phosphate groups. Forexample, the second composition can comprise polyvinylphosphonic acid;2-carboxyethyl phosphonic acid; amino tri (methylene phosphonic acid);1-hydroxyethylidene-1,1,-diphosphonic acid; hexamethylenediaminetetra(methylenephosphonic acid); diethylenetriamine penta(methylenephosphonic acid); ethylenediamine tetra(methylene phosphonic acid);bis(hexamethylene triamine penta(methylenephosphonic acid));2-phosphonobutane-1,2,4-tricarboxylic acid; or monoethanloaminediphosphonate. As additional examples, the second composition cancomprise an alkyl phosphate or an alkyl phosphonate, such as octylphosphonic acid, decyl phosphonic acid, dodecyl phosphonic acid,hexadecyl phosphonic acid, octadecyl phosphonic acid, docosyl phosphonicacid, hydroxy-dodecyl phosphonic acid, hydroxy-undecenyl-phosphonicacid, decanediylbis(phosphonic acid), dodecylphosphate, orhydroxy-dodecylphosphate.

The two surface components can be differentially modified by differentcompositions. Thus, in one class of embodiments, the methods includecontacting the substrate with a first composition that selectivelyassociates with the first surface component and coupling the molecule ofinterest to the first composition to selectively couple the molecule ofinterest to the first surface component, and contacting the substratewith a second composition that selectively associates with the secondsurface component (before or after coupling of the molecule of interestor deposition of the first composition).

In an alternative aspect, the invention provides methods of selectivelydepositing a molecule of interest on selected regions of a substrate,comprising providing a substrate having first and second components, thefirst component comprising a conductive material and the secondcomponent comprising an insulator, and applying an electrical potentialto the first component so as to increase or decrease association of amolecule of interest with a surface of the first component.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a schematic illustration of a Zero Mode Waveguide (ZMW) inapplication.

FIG. 2 provides a schematic illustration of a light directed surfaceactivation process of the invention.

FIG. 3 provides a schematic illustration of a process for providingactive surfaces in optically relevant portions of optical confinementslike ZMWs.

FIG. 4 provides a simulated plot of surface activation level as afunction of the distance from the bottom surface of a ZMW over twoseparate activation stages.

FIG. 5 provides a schematic illustration of an alternate lightactivation strategy using a two activation step process.

FIG. 6 provides a schematic illustration of a diffusion limited processfor providing active surfaces within confined structures.

FIG. 7 provides an illustration of process for providing a printedmasking layer on non-relevant surfaces of substrates.

FIG. 8 schematically illustrates a photocleaving process for removingactive groups from non-relevant portions of substrate surfaces.

FIG. 9 illustrates a size excluded particle based process for removingmolecules of interest from non-relevant portions of substrate surfaces.

FIG. 10 illustrates selective immobilization of molecules of interestusing an electrically driven system.

FIG. 11 schematically illustrates a process for removal of moleculesfrom non-relevant surfaces of substrates using an entraining matrixfollowed by a lift-off technique.

FIG. 12 illustrates the effects of selective immobilization processes ofthe invention and particularly using a size excluded particle process.

FIG. 13 schematically illustrates a process for selective localizationof molecules using an alternate exclusionary process.

FIGS. 14A and 14B schematically illustrate an exemplary process forselective localization of molecules using an exclusionary process inwhich a site-specific deactivation component removes the molecule ofinterest from the substrate.

FIGS. 15A, 15B and 15C schematically illustrate an exemplary process forselective localization of molecules using an exclusionary process inwhich a site-specific deactivation component removes a coupling moietyfrom the substrate.

FIG. 16 schematically illustrates selective immobilization of moleculesof interest by exploiting differing surface characteristics of differentmaterials in hybrid substrates like ZMWs and passivation with a PE-PEGcopolymer.

FIG. 17 schematically illustrates formation of a polyelectrolytemultilayer.

FIG. 18 schematically illustrates selective immobilization of moleculesof interest by exploiting differing surface characteristics of differentmaterials in hybrid substrates like ZMWs and passivation with apolyelectrolyte multilayer.

FIG. 19 illustrates binding of nucleotide analogs to a polyelectrolytemultilayer-treated versus a plasma-PDMS treated (non-biased treated)surface.

FIGS. 20A and 20B illustrate binding of polymerase to a polyelectrolytemultilayer-treated versus an untreated aluminum surface.

FIG. 21 illustrates the effects of selective immobilization processes ofthe invention and particularly using a selective silanization andpolyelectrolyte multilayer passivation process.

FIG. 22 illustrates the effects of selective immobilization processes ofthe invention and particularly using a selective silanization andpolyelectrolyte multilayer passivation process.

FIG. 23 illustrates binding of neutravidin-coated fluorescent beads to aphosphonate-treated ZMW versus an untreated ZMW.

FIG. 24 illustrates binding of nucleotide analogs to aphosphonate-treated ZMW versus an untreated ZMW.

Schematic figures are not necessarily to scale.

DETAILED DESCRIPTION OF THE INVENTION I. GENERAL DESCRIPTION OFINVENTION

The present invention is generally directed to methods and processes forproviding desired molecules in preselected locations or areas on asubstrate or within a set volume, and articles made from such methods orprocesses, and particularly, in desired low concentrations or asindividual molecules, within an optical confinement. In particularlypreferred aspects, the invention is directed to methods for localizingindividual molecules within a particular space or volume, such that thespatial individuality of the molecule may be exploited, e.g.,chemically, optically, electrically, or the like. The invention alsoprovides the substrates, devices, receptacles and the like, e.g., theoptical confinements, produced by these processes. While the processesof the invention may be broadly practical in providing individualmolecules within any of a variety of given desired space or volumetypes, in particularly preferred aspects, the processes are used toselectively deposit or immobilize a desired molecule, such as an enzyme,within the optically accessible portion of an optical confinement, andparticularly, a zero mode waveguide (ZMW).

In general, optical confinements are used to provide electromagneticradiation to or derive such radiation from only very small spaces orvolumes. Such optical confinements may comprise structural confinements,e.g., wells, recesses, conduits, or the like, or they may compriseoptical processes in conjunction with other components, to provideillumination to or derive emitted radiation from only very smallvolumes. Examples of such optical confinements include systems thatutilize, e.g., total internal reflection (TIR) based optical systemswhereby light is directed through a transparent substrate at an anglethat yields total internal reflection within the substrate.Notwithstanding the TIR, some small fraction of the light will penetratebeyond the outer surface of the substrate and decay rapidly as afunction of distance from the substrate surface, resulting inillumination of very small volumes at the surface. Similarly, ZMWstructures may be employed that utilize a narrow core, e.g., from 10 to100 nm, disposed through a cladding layer where the core is dimensionedsuch that the desired electromagnetic radiation is prevented frompropagating through the core. As a result, the radiation will permeatethe core only a very short distance from the opening of the core, andconsequently illuminate only a very small volume within the core. Avariety of other optical confinement techniques, including, e.g., fieldenhancement by sharp metal tips, nanotube confinement, thin slitconfinement, near-field resonant energy transfer confinement, near fieldaperture confinement, diffraction limited optical confinement, andstimulated emission depletion confinement, are contemplated, as well asall other confinements described in pending U.S. Ser. No. 10/944,106 andSer. No. 09/572,530 and U.S. Pat. No. 6,917,726, each of which isincorporated herein by reference in its entirety for all purposes.

Zero mode waveguides (ZMWs) are generally characterized by the existenceof a core surrounded by a cladding, where the core is dimensioned suchthat it precludes a substantial amount of electromagnetic radiation thatis above a cut-off frequency from propagating through the core. As aresult, when illuminated with light of a frequency below the cutofffrequency, the light will only penetrate a short distance into the core,effectively illuminating only a small fraction of the core's volume. Inaccordance with the present invention, the core comprises an empty orpreferably fluid filled cavity surrounded by the cladding layer. Thiscore then provides a zone or volume in which a chemical, biochemical,and/or biological reaction may take place that is characterized byhaving an extremely small volume, and in some cases sufficient toinclude only a single molecule or set of reacting molecules. ZMWs, theirfabrication, structure, and use in analytical operations are describedin detail in U.S. Pat. No. 6,917,726 and Levene, et al., Science299(5607):609-764 (2003), the full disclosures of which are herebyincorporated herein by reference in their entirety for all purposes.

In the context of chemical or biochemical analyses within ZMWs as wellas other optical confinements, it is clearly desirable to ensure thatthe reactions of interest are taking place within the opticallyinterrogated portions of the confinement, at a minimum, and preferablysuch that only a single reaction is occurring within an interrogatedportion of an individual confinement. A number of methods may generallybe used to provide individual molecules within the observation volume. Avariety of these are described in co-pending U.S. patent applicationSer. No. 11/240,662, filed Sep. 30, 2005, incorporated herein byreference in its entirety for all purposes, which describes, inter alia,modified surfaces that are designed to immobilize individual moleculesto the surface at a desired density, such that approximately one, two,three or some other select number of molecules would be expected to fallwithin a given observation volume. Typically, such methods utilizedilution techniques to provide relatively low densities of couplinggroups on a surface, either through dilution of such groups on thesurface or dilution of intermediate or final coupling groups thatinteract with the molecules of interest, or combinations of these.

In some cases, it may be further desirable that reactions of interest bereduced or even eliminated from other regions outside of the observationvolume, e.g., on the overall substrate housing ZMWs, the cladding layer,etc., both inside and outside of the observation volume. In particular,reactions that are outside of the range of interrogation may,nonetheless, impact the reaction of interest or the monitoring of thatreaction, by affecting reaction kinetics through depletion of reagents,increasing concentration of products, contributing to signal backgroundnoise levels, e.g., through the generation of products or consumption ofreactants, that may interfere with the interrogated reaction or thatprovide excessive detectable background product levels that diffuse intoand out of the interrogation volume of the waveguide. Accordingly,selective and preferential deposition and/or immobilization of thereaction components within the observation volume are particularadvantages of the invention. These are generally practicable both as analternative to and, preferably, in addition to the low densitydeposition methods referenced above. In the context of the foregoing,molecules of interest may be described as being preferentially locatedin a particular region, or localized substantially in a given region. Itwill be appreciated that use of the term preferentially is meant toindicate that the molecule is localized in a given location at aconcentration or surface density that exceeds that of other locations inwhich it is not preferentially localized. Thus preferentialimmobilization of a given molecule in a first region will mean that themolecule is present in such region at a higher density or concentrationthan in other regions. Density in such regions may be as much as 20%greater, 30% greater, 50% greater, 100% greater, or upwards of 200%, upto 1000% or more of the concentration or density in other regions, andin some cases 100 times greater, 1000 times greater or more. Similarmeaning is generally applicable to indications that a given molecule issubstantially only located in a given region.

In the case of, for example, ZMWs used for single molecule enzymaticanalysis, it may be desirable to provide a single enzyme molecule withinthe illumination volume of a waveguide, and preferably upon the bottomor base surface of the waveguide. As noted above, it may therefore befurther desirable to ensure that additional enzyme molecules are notpresent upon surfaces other than the bottom surface, e.g., the walls ofthe core and/or the surfaces of the cladding layer that are not part ofthe core, and the like.

A particularly valuable application of the substrates produced by theprocess of the invention is in processes termed “single moleculesequencing applications.” By way of example, a complex of a templatenucleic acid, a primer sequence and a polymerase enzyme may bemonitored, on a single molecule basis, to observe incorporation of eachadditional nucleotide during template dependent synthesis of the nascentstrand. By identifying each added base, one can identify thecomplementary base in the template, and thus read off the sequenceinformation for that template. In the context of ZMWs, an individualpolymerase/template/primer complex may be provided within theobservation volume of the ZMW. As each of four labeled (e.g.,fluorescent) nucleotides or nucleotide analogs is incorporated into thesynthesizing strand, the prolonged presence of the label on suchnucleotide or nucleotide analogs will be observable by an associatedoptical detection system. Such sequencing processes and detectionsystems are described in, e.g., Published U.S. Patent Application No.2003/0044781 and pending U.S. patent application Ser. No. 11/201,768,filed Aug. 11, 2005, the full disclosures of which are incorporatedherein by reference in their entirety for all purposes. Such singlemolecule sequencing applications are envisioned as being benefited bythe methods described herein, through the selected immobilization ofpolymerases, templates or primers or complexes of any or all of these,preferentially within selected regions on a substrate, and/orsubstantially not on other portions of the substrate.

In general, selective provision of a molecule of interest in a givenlocation, e.g., in the illumination volume within a ZMW, may beaccomplished using either additive or subtractive processes. By additiveprocess, is generally meant that the individual molecule is placed ordeposited in the desired location and not elsewhere. By contrast,subtractive processes denote the deposition of the molecule of interestmore ubiquitously and non-selectively, e.g., over an entire substratesurface, followed by the selected removal of the molecule of interestfrom the non-desired locations. While these descriptions provideconvenience in describing various processes, it will be appreciated thatthe result of one process may be indistinguishable from the result ofthe other process. It will also be appreciated that many processes mayinclude steps that may be described as either additive, subtractive, orboth. Although generally discussed in terms of localization of enzymesor other macromolecular groups, for purposes of the present invention,the molecule of interest may be any of a variety of different functionalmolecules for which one desires to provide spatial individuality orenhanced localization. Such groups include active molecules, such ascatalytic molecules like enzymes, but also include molecules with morepassive functionality, e.g., non catalytic groups, such as binding orcoupling groups, hydrophobic or hydrophilic groups, structuralenhancement groups, e.g., for adhesion promotion, activatable ordeactivatable groups, or the like. Binding or coupling groups mayinclude small molecule coupling groups or they may includemacromolecular coupling groups, e.g., antibodies, antibody fragments,specific binding pairs, such as avidin/biotin, binding peptides,lectins, complementary nucleic acids, or any of a variety of otherbinding groups. Catalytically active molecules will typically includeany catalytically active molecule for which one desires spatialindividuality, e.g., to exploit in single molecule analyses, or thelike.

In at least one aspect, the present invention is directed to providingenhanced isolation of discrete reaction and/or observation regions. Thisis not simply to provide optical isolation between such regions, butalso to provide chemical and/or environmental isolation for suchregions. In a general sense, this is accomplished by providing a barrieror zone between reaction and/or observation regions that substantiallyprevents the diffusion of reactants and/or products from outside aparticular reaction zone from entering and potentially interfering withthe reaction taking place therein, or the observation of that reaction.In providing the requisite isolation, one may focus on one or both of:(1) providing sufficient separation/isolation between neighboringreaction/observation regions; and (2) eliminating any potentiallyinterfering components from the spaces between such neighboring regions,e.g., clearing any reactants, products and/or enzymes from such spaces,and creating a type of “demilitarized zone” between observation regions.

Providing enhanced isolation generally relates to providing a barrier ofsome sort between observation regions. In general, such barriers maysimply include sufficient distance in a fluidic system such thatreactants and products may not diffuse from one reaction into aparticular observation region, whether the reaction is in a neighboringobservation region or is located somewhere else. One may provide suchdistance across a planar substrate or one may increase the effectivediffusion distance by providing a structured or contoured surface on thesubstrate. For example, in particularly preferred aspects, one mayprovide discrete reaction/observation regions within nanoscale wells toeffectively increase the distance between such regions, as well as treator otherwise produce such substrates, to reduce or eliminate anyreactants and/or products from existing or being generated in the spaceor regions between the selected regions, e.g., surfaces other than thoseat or toward the bottom surface of the nanoscale wells.

II. ADDITIVE PROCESSES

As noted above, in at least one aspect, an additive process is employedto provide the desired immobilized molecules of the invention. Theadditive processes typically rely upon the selective provision ofbinding or coupling groups at the desired location, followed by thedeposition of the molecules of interest. This deposition may, again, bethe result of additive or subtractive processes.

In at least a first aspect, the additive processes of the inventiontypically include the deposition of a coupling group upon the substratesurface that selectively binds the molecule of interest only within thedesired region on the surface, e.g., within the observation area of anoptical confinement such as a ZMW. Coupling of functional groups,including activatable functional groups, to surfaces may generally becarried out by any of a variety of methods known in the art. Forexample, in the context of silica based substrates, e.g., glass, quartz,fused silica, silicon, or the like, well characterized silanechemistries may be used to couple other groups to the surface. Suchother groups may include functional groups, activatable groups, and/orlinker molecules to either of the foregoing, or the actual molecules ofinterest that are intended for use in the end application of thesurface. In the context of other substrate types, e.g., polymericmaterials, metals or the like, other processes may be employed, e.g.,using hybrid polymer surfaces having functional groups coupled theretoor extending from the polymer surface using, e.g., copolymers withfunctional groups coupled thereto, metal associative groups, i.e.,chelators, thiols, or the like.

In at least a first aspect of the invention, providing coupling of amolecule of interest only within a desired area or region is typicallycarried out by providing an activatable coupling group coupled to thesurface of the overall substrate that is selectively activated onlywithin the desired region, or by using a selectively de-activatablecoupling group and selectively deactivating it in all but the desiredregion. The selective provision of active coupling groups only wheredesired allows selective deposition and coupling of the molecule ofinterest substantially only in the desired regions. For ease ofdiscussion, the portion of a surface or substrate in which one wishes toselectively provide molecules of interest for a given application arereferred to herein as the “desired regions” while regions outside ofthese regions are referred to as the non-desired regions. Such desiredand non-desired regions may include planar surfaces or may comprisethree dimensional structures such as wells, recesses, surfaceirregularities, posts, pillars, trenches, troughs, channels,capillaries, porous materials, or the like.

A variety of different activatable coupling groups may be used inconjunction with this aspect of the invention. Typically, such groupsinclude coupling groups that are capped or blocked with a selectivelyremovable group. These include groups that are thermally altered, e.g.,thermolabile protecting groups, chemically altered groups, e.g., acid orbase labile protecting groups, and photo alterable groups, e.g.,photo-cleavable or removable protecting groups.

Deactivation of coupling groups, e.g., in non-desired regions, maycomprise the use of groups that may be directly selectively deactivated,e.g., through the use of thermal, chemical or photo-induced chemistriesthat cap or result in the removal of functional groups, i.e., throughphoto-induced cross-linking, photocleavage, or the like. Alternatively,and in certain preferred aspects, such deactivation methods utilizeselective activation of the coupling group in the non-desired regions,followed by blocking or capping of the resulting active coupling groupwith a neutral or inert blocking group, e.g., a group that issubstantially incapable of coupling to the molecule of interest, or anintermediate linking molecule, under coupling conditions subsequentlyapplied to couple such groups to the desired regions. This subsequentlyadded blocking group may be irreversible or reversible. However,reversibility of such capping, if any, will typically involve amechanism other than that of the underlying activatable coupling group,to avoid re-activating capped groups in the non-desired regions whileactivating those underlying activatable groups in the desired regions.For example, where one is employing a photoactivation strategy toselectively activate groups in the desired regions, capping groupsapplied to non-desired regions will typically not be photoactivatable orotherwise activated by any conditions to which the surface will beexposed in application.

Following the capping of coupling groups in the non-desired regions, thecoupling groups within the desired regions, or area of interest, may beselectively activated and coupled with the molecule of interest. Forease of discussion, whether photoactivation involves photocleavage of ablocking group, or photoactivation through alteration of a chemicalstructure without removal of a larger blocking group, per se, e.g.,results in modified groups or addition of other groups, it willgenerally be referred to herein as activation, e.g., photoactivation.

In at least one particularly preferred aspect, photoactivatable couplinggroups are used to selectively deposit molecules of interest in desiredregions, e.g., using chemically active coupling groups that are cappedwith a photo-labile protecting groups. Such photoactivatable couplingmechanisms are particularly useful for systems that employ opticalconfinements such that light for both observation of an ultimatereaction of interest and for activation of the coupling group is onlycapable of illuminating the desired region, e.g., those regions of a ZMWclosest to the core opening from which the core is illuminated. Inparticular, because activating light directed at a ZMW will onlyilluminate a restricted volume, e.g., the illumination volume, moleculesof interest will be selectively coupled substantially only within theillumination volume. Restated, the same optical confinement effect usedto only monitor reactions within the small confined volume of theillumination volume (which typically substantially defines theobservation volume in the applicable analytical operations to which theZMW will be put), likewise only permits activation (and consequentcoupling) within that same confined volume or portion of the ZMW. Aswill be appreciated, by modulating the activation radiation, one canfurther control the illumination volume during activation to be asmaller volume than the illumination volume during application. Inparticular, by applying a lower power illumination, using a longerwavelength of activation light than illumination/interrogation light,one can illuminate, activate and thus couple molecules of interest onlyto a subset of the surface that will ultimately be within theillumination volume in the ultimate application.

For a number of the specific aspects of the invention, it is generallypreferred to utilize a substrate that provides for the selectivedirection of electromagnetic radiation to desired regions, both in termsof the ultimate application of such substrates, e.g., in interrogatingchemical, biochemical and/or biological reactions on those substrates,and in providing selectively activated surfaces for selectivelyimmobilizing molecules of interest in those regions for exploitationduring such analyses. In sum, one takes a basic function of thesubstrate that is used in its ultimate application, and exploits thatfunction to improve the fabrication and processing of that substrate forthat application. In the context of directing radiation, a substratethat is used to focus radiation into desired regions for interrogationof reactions within such regions is processed using the same radiationdirecting properties to selectively functionalize those desired regions.

A variety of different coupling groups may be used in this context,depending upon the nature of the molecule of interest to be subsequentlydeposited upon and coupled to the substrate. For example, the couplinggroups may include functional chemical moieties, such as amine groups,carboxyl groups, hydroxyl groups, sulfhydryl groups, metals, chelators,and the like. Alternatively or additionally, they may include specificbinding elements, such as biotin, avidin, streptavidin, neutravidin,lectins, SNAP-tags™ or substrates therefore (Covalys Biosciences AG; theSNAP-tag™ is a polypeptide based on mammalianO6-alkylguanine-DNA-alkyltransferase, and SNAP-tag substrates arederivates of benzyl purines and pyrimidines), associative or bindingpeptides or proteins, antibodies or antibody fragments, nucleic acids ornucleic acid analogs, or the like. Additionally, or alternatively, thecoupling group may be used to couple an additional group that is used tocouple or bind with the molecule of interest, which may, in some casesinclude both chemical functional groups and specific binding elements.By way of example, a photoactivatable coupling group, e.g.,photoactivatable biotin, may be deposited upon a substrate surface andselectively activated in a given area. An intermediate binding agent,e.g., streptavidin, may then be coupled to the first coupling group. Themolecule of interest, which in this particular example would bebiotinylated, is then coupled to the streptavidin.

Photo-labile protecting groups employed in this aspect of the inventionmay include a variety of known photo-cleavable protecting groups,including, for example, nitroveratryl, 1-pyrenylmethyl,6-nitroveratryloxycarbonyl, dimethyldimethoxybenzyloxycarbonyl,2-nitrobenzyloxycarbonyl, methyl, methyl-6-nitropiperonyloxycarbonyl,2-oxymethylene anthraquinone, dimethoxybenzyloxy carbonyl,5-bromo-7-nitroindolinyl, o-hydroxy-alpha-methyl cinnamoyl, and mixturesthereof, as described in U.S. Pat. No. 5,412,087 and 5,143,854, each ofwhich is incorporated herein by reference in its entirety for allpurposes.

Coupling of the photoactivatable coupling groups to the surfaces ofinterest may be accomplished by a number of methods known in the art.For example, photoprotected or activatable groups may include a carboxylgroup that is coupled through hydroxyl groups on the surface or attachedto the surface through a linker group, e.g., a PEG molecule.Alternatively, amine groups on the photoactivatable groups may becoupled to surface bound epoxy groups. Alternatively, activatable groupsprecoupled to linker molecules, e.g., PEG groups, may be silanated andattached directly to surfaces through known processes.

Examples of the compounds used in the foregoing coupling strategies,e.g., using MeNPOC protected biotin, are illustrated below:

Additional light sensitive protecting groups include groups useful forcoupling amines, such as trimethylphenyloxycarbonyl (TMPOC), groupsuseful for coupling acids, such as phenacyl ester (313 nm cleavage),α-phenacyl ester, Desyl ester (350 nm), Bis(o-nitrophenyl)methyl ester(320 nm), 1-pyrenylmethylester(340 nm),N-8-nitro-1,2,3,4-tetrahydroquinolylamide (350 nm), as well as esters ofthe following compounds:

For those aspects of the invention that use longer wavelengths foractivation or deprotection, appropriate longer wavelength labile groupswould be used, such as brominated 7-hydroxyxoumarin-4yl-methyls, whichare photolabile at around 740 nm. Other such groups are known to thoseof skill in the art.

Also useful are such photolabile groups for coupling to alcohols,including, e.g., some of the groups described above, as well asp-nitrobenzyloxymethyl ether, p-methoxybenzylether, p-nitrobenzylether,mono, di or trimethoxytrityls, diphenylmethylsilyl ether, sisyl ether,3′,5′-dimethoxybenzoincarbonate, methanesulfate, tosylate, and the like.These and a variety of other photocleavable groups may be employed inconjunction with this aspect of the invention, and are described in,e.g., the CRC Handbook of Organic Photochemistry and Photobiology,Second Edition, and Protective Groups in Organic Synthesis (T. W. Greeneand P. G. Wuts, 3^(rd) Ed. John Wiley & Sons, 1999), each of which isincorporated herein by reference in its entirety for all purposes.

In addition to, or as an alternative to, the use of the previouslydescribed, relatively large, photo-removable protecting groups, theinvention also includes the use of photoactivatable groups, e.g., groupsthat are chemically altered, other than through the removal of suchblocking groups. For example, vinyl or allyl groups may be coupled tosurfaces and simultaneously illuminated and coupled with appropriategroups to be coupled that bear, e.g., sulfhydryl groups, such as biotinhaving a sulfhydryl group coupled to it either directly or through alinker molecule, which react with the activated vinyl or allyl group tocouple to the surface. Alternatively, other groups, like nitroarylazidesmay be employed as the activatable coupling groups. A wide variety ofother photoactivatable compounds may likewise be used, including, e.g.,nitrospiropyran groups (See, Blonder et al., J. Am. Chem. Soc. 1997,119:10467-10478, and Blonder et al., J. Am. Chem. Soc. 1997,119:11747-11757.

In one aspect, a photoinitiator, e.g., a long wavelength photoinitiator,is employed, such as Irgacure 784 (bis-(eta5-2,4-cyclopentadien-1-yl)bis[2,6-difluoro-3-(1H-pyrrol-1-yl)phenyl]titanium;Ciba Specialty Chemicals) that can initiate free radical reactions atwavelengths as long as 530 nm. Such long wavelength photoinitiators havea variety of applications. For example, a surface (e.g., a metal oxidesurface) can be coated with vinyl-alkyl-phosphonate. Exposure of adesired region of the surface to a 530 nm laser in the presence ofIrgacure 784 and biotin-PEG-SH results in formation ofbiotin-PEG-alkyl-phosphonate in that region. The biotin can subsequentlybe employed to immobilize a molecule of interest to the desired region.

In related aspects, the photoactivatable component may be provided insolution and activated proximal to the surface region where localizationis desired. For example, one may graft an activatable binding componentor other molecule of interest onto an active silane surface. One exampleof such a system includes photoactivatable psoralen-biotin compounds(available from, e.g., Ambion, Inc.), that are activatable under UVlight for coupling with a silanated surface, e.g., a trimethoxysilanemodified surface.

Those aspects of the invention that include an additive process using aselective surface activation generally encompass a number of differentstrategies for selective activation in the desired locations. Suchstrategies may include a single activation step, a multiple activationstep process, a multiple step process that includes both activation anddeactivation steps or processes, or the like. For ease of discussion,such multiple step processes are described with reference tophotoactivation and/or photodeactivation processes, although it will beappreciated that other non-photo driven processes may be similarlyemployed.

In at least a first, relatively simple aspect, the selective activationof photo-activatable coupling groups in the desired region involves asingle step of directing activating radiation at the desired region andcoupling the molecule of interest to the activated coupling groups thatare disposed thereon. As noted, in the case of optical confinementswhere it is desirable to localize the molecule of interest, e.g., anenzyme, within the illumination volume, the single step photo-drivenactivation should result in coupling substantially only within theillumination volume. Further, as noted previously, by modulating theactivation radiation, one can further focus the activation, and thuscoupling of groups of interest, in a subset of the illumination volumethat is interrogated during the ultimate application, e.g., in nucleicacid sequence determination using an immobilized polymerase enzyme.

The basic functional structure of a ZMW structure is schematicallyillustrated in FIG. 1. As shown, a ZMW structure 100 is provided thatincludes a cladding layer 102 deposited upon a transparent substratelayer 104. A core 106 is disposed through the cladding layer to exposethe transparent layer 104 below. The core is dimensioned to provideoptical confinement by preventing propagation of electromagneticradiation that falls below a cut-off frequency through the core.Instead, the light only penetrates a short distance into the core,illuminating a relatively small volume, indicated as bounded by thedashed line 108. By providing reactants of interest within theobservation volume, e.g., enzyme 110 and substrate 112, one canselectively observe their operation without interference from reactants,e.g., substrates 114 outside the observation volume, e.g., above line108.

As noted previously, it is generally desirable that in performingmolecular analyses, e.g., enzyme analyses, that the molecule of interestbe provided preferentially within the illumination or observationvolume. Accordingly, a simple activation strategy, as applied to ZMWs,is schematically illustrated in FIG. 2, with reference to FIG. 1. Asshown, the ZMW structure 100 may be first treated to provide anactivatable surface, e.g., shown as solid line 202. As shown, thetreatment step is not selective, in that it provides such an activatablesurface over the entire surface of the structure, including claddinglayer 102. The activatable groups that are within the illuminationvolume, e.g., as bounded by dashed line 108, are then subjected toactivation (as indicated by dashed line 204). In the context of a ZMWstructure, this typically involves exposing the activatable groups toactivating radiation through the transparent substrate 104, as indicatedby waved arrows 206. As will be appreciated, the activation radiationdecays sufficiently beyond the illumination volume, and as such,substantially activates only the groups therein, e.g., those belowdashed line 108. Molecules of interest, e.g., enzymes, or enzymespecific coupling groups, are then coupled to the activated groupswithin the observation volume, and nowhere else on the surface. It willbe appreciated that the reference to the illumination volume as having awell defined border is simplified for ease of discussion, and that decayof illumination through the ZMW core is not so abrupt. As a result, boththe illumination and, as a result, the level of surface photo-activationfrom such illumination would be expected to decrease in a relatedfashion with increasing distance from the illuminated end of thewaveguide core. The rate of radiation decay and the activation levelsmay decrease at different rates, depending upon the nature of theactivation processes, e.g., whether there is saturation at any point, aswell as whether the activation processes are single or multiple photonprocesses.

In an alternative process, an additional activation step may be employedto further select the region to which molecules of interest may becoupled. In particular, in a given activation step within an opticalconfinement, e.g., a ZMW, illumination as shown in FIGS. 1 and 2 willgenerally result in a spectrum of activation within the confinement,with more activated groups being present where illumination is greatest,e.g., at the bottom surface of the waveguide. As the illuminationdecreases with further penetration into the waveguide, the activationlevel or efficiency of activation will decrease depending upon thecharacteristics of the activatable group the intensity of theillumination and the amount of time exposed. This will result in adecreasing probability of activation of groups in the portions of theillumination region where light penetration decreases and thus,illumination is less. By then capping these activated groups with asecond photoremovable group and repeating the activation step, theprobability of the groups present being activated away from highillumination is similarly limited, but now is applied to a smallernumber of groups. This is further illustrated with the followingexample: if one has a uniform distribution of photoactivatable groups ina ZMW structure that are activatable with a first wavelength of light,at a particular distance from the bottom of the waveguide, one half ofall activatable groups present are activated. If the active groups arethen capped with a second photoactivatable group that is activated at adifferent wavelength, activation of those groups will again activateonly half of the activatable groups present at the particular distance,or one fourth of the originally activatable groups. The result whenapplied over the spectrum of activation is a more narrowly focusedactivation/coupling area approaching the bottom of the waveguidestructure.

A schematic illustration of a double activation method is provided inFIG. 3. In accordance with the double activation method, a waveguidestructure 300, for example, is provided with a surface coating ofphotoactivatable groups uniformly applied over the surface (shown inpanel I, as black diamonds 302). A first activation step (panel II) isused to activate the activatable groups within a waveguide (shown asopen diamonds 304) by, e.g., directing an activation light through thebottom surface 306 of the waveguide 300. Instead of coupling themolecule of interest to those activated groups, a second activatablegroup (shown as black circles 308 in Panel III), that is activated by adifferent wavelength of light can be used to cap the activated groups304. A subsequent activation step (Panel IV) then activates a subset ofthe newly capped groups (shown as open circles 310), and the molecule ofinterest (not shown) is then coupled to these newly activated groups.FIG. 4 provides an exemplary simulated plot of surface activation(concentration of activated surface groups) vs. distance from the bottomsurface of a ZMW, for both a first and second activation step. As shown,a first activation step would be expected to yield an activation profilethat falls off in conjunction with a rate of decay of activation lightaway from the bottom surface of a ZMW. After capping with a secondphoto-removable group, and reactivation at a different wavelength, onewould expect a similar decay profile, but based upon only the previouslyactivated groups. As a result, the activated groups would be morefocused at the bottom surface of the waveguide than with just a singleactivation step. While described in terms of two steps, it will beappreciated that more steps could be performed to further focus theactivated region on the surface.

As used herein, unless indicated otherwise from the specific context,capping generally refers to coupling an additional group to an otherwisereactive group such that the resulting compound is not active to furtherapplied coupling or other reactions of interest. Such capping moleculestypically comprise groups that will couple to the exposed coupling groupbut which are otherwise natural to the desired reaction, and will varydepending upon the nature of the groups to be capped. They may includeneutral silane groups for capping silanol surface groups, or they mayinclude other non-reactive materials, e.g., non-reactive organicmaterials, e.g., alcohols, alkyl groups, alkenyl groups, or the like.Such capping groups may be small molecules or may include largerpolymeric or macromolecular structures, such as polyethylene glycols(PEGs), or the like. Capping chemistries are widely practiced in surfacemodification, derivatization and passivation processes that arediscussed in, e.g., Immobilized Biomolecules in Analysis: A PracticalApproach (Cass and Ligler Eds.) Oxford University Press, 1998, andHermansonn et al , Immobilized Affinity Ligand Techniques, AcademicPress, Inc. 1992, the full disclosures of which are incorporated hereinby reference in their entirety for all purposes.

In another multi-step approach, iterative steps of activation anddeactivation may be employed to focus the coupling of the molecule ofinterest. As noted previously, photoactivatable groups may be employedin accordance with the deactivation schemes described above, e.g., whereareas other than the desired area are activated and capped or blocked,followed by activation within the area of interest and coupling of themolecule of interest. This method may prove more useful for applicationsbased upon ZMWs. In particular, through an illumination from the openend of the waveguide, one will typically activate, and subsequently capactivatable groups not only on the upper surface of the cladding layer,but also, some portion of the activatable groups on the walls of thewaveguide core. Subsequent activation from the bottom or closed end ofthe core will then only be able to activate those activatable groupsthat have not yet been capped. To the extent activation radiationpenetrates greater than half the length of the core; this will result ina greater selection of activation for deposition at or toward the bottomof the ZMW. Such a method is schematically illustrated in FIG. 5.

In particular, on a substrate having optical confinements, such as ZMW500, disposed upon it, one can provide a uniform surface that includesphoto-activatable coupling groups (filled diamonds 502) over the entiresurface, e.g., inside and outside of the confinement (Step I). In asubsequent step (step II), the surface is exposed to activationradiation from a top side, e.g., the side away from the area where onewishes to immobilize the molecules of interest. The activated groups(open diamonds 504) are then inactivated (Step III) by capping them withanother protecting group (filled circles 506), e.g., a non-removableprotecting group. Subsequently, the ZMWs are illuminated from thebottom, so that the illumination volume includes the desired regions andcoupling groups in that region are activated (Step IV, open diamonds508). The molecules of interest are then coupled to these activatedgroups. By controlling the initial activation illumination, one caneffectively control the amount of activatable groups that are cappedprior to the later activation step. In particular, by using activationradiation, or a waveguide geometry or other exposure conditions, thatpermit activation radiation to effectively propagate more than half waythrough the core of the waveguide, in the first activation step, one mayeffectively cap more than half of the activatable groups in the firstactivation and capping step. By then directing activation radiation fromthe bottom side, substantially all of the remaining activatable groups,which are primarily substantially disposed toward the bottom of the corewhich would not have been activated and capped in the first steps, maythen be activated and made available for coupling to the molecules ofinterest. As will be appreciated, the various approaches described abovemay be combined to further enhance selectivity.

In an alternative process schematically similar to the photoactivationmethods described above, deep UV etching processes may be employed ingenerating an active surface in desired regions, e.g., at the bottomsurface of a ZMW. In particular, deep UV exposure, e.g., illumination atbelow 200 nm, i.e., using deep UV lasers, deep UV lamps, e.g., Xeradexexcimer lamp, under vacuum has been used to selectively degrade surfacebound organic or inorganic materials, as such UV exposure is capable ofbreaking chemical bonds directly, e.g., without assistance from oxygenradicals which may be formed during the process, which may contribute toexcessive etching. By performing such exposure under vacuum or otherrestrictions on the ability of oxygen radicals to contact and etch othersurfaces, one can irradiate and consequently controllably remove organicand inorganic materials from selected substrate regions.

In the context of the surfaces of the invention, for example, a ZMWsubstrate may be provided with a first blocking layer that issubstantially inert to additional coupling groups, e.g., it isnon-reactive with the coupling strategy to be employed in eventuallyjoining the molecules of interest to the surface. As a result, thefunctional groups on the original surface are effectively blocked bythis blocking layer. Examples of blocking layers include organosilanes,such as PEG-silane, deep UV resists, or other long chain organosilanes.Exposure of the waveguides from the bottom or substrate side to deep UVradiation then degrades the blocking layer within the waveguides andpreferentially at the bottom surface of the waveguide.

During the exposure or etching process, it may be desirable to limit theability for oxygen radicals to contact other portions of the surface,e.g., outside of the ZMW or outside the observation region toward thebottom of the ZMW. In such cases, the system may be operated undervacuum, or alternatively or additionally, a sealing layer may beprovided over the ZMW. Such sealing layer may comprise a rigid layer,e.g., a glass or silicon wafer or a more flexible material, such as apolymer sheet, e.g., PDMS, PTFE, polypropylene, polyethylene,polystyrene, or any of a variety of polymeric materials that are capableof sealing the waveguide structures, preferably without excessiveoff-gassing or otherwise contributing undesired chemical residues to thewaveguides.

Following exposure, the substrate is contacted with a material thatincludes the functional groups used to couple the molecule of interest,which binds preferentially to the unblocked region, e.g., the exposedsilanol groups uncovered by the ‘etching’ process. This additionalmaterial may include only functionalized groups or it may include amixture of functionalized and inert groups in order to control densityof functional groups, and consequently, molecules of interest within thewaveguide structure. Such functionalized groups may be reactive chemicalspecies and/or specific binding moieties, such as avidin, biotin, or thelike.

Once the appropriate density of coupling groups is deposited in thedesired regions, e.g., at the bottom surface of the waveguide structure,the molecule of interest may be coupled to the coupling groups, e.g.,through the reactive group or through an appended biotin or avidin groupor other specific binding partner to the coupling group or that islinked to the coupling group.

In another process similar to the photoactivation methods describedabove, tethered or grafted photoinitiators are employed. Of particularinterest are photo-iniferters such as dithiocarbamates (DTC) whichinitiate and control the radical polymerization of acrylates, alkenes orthe terminal radical addition of a capping reagent with a ligand forspecific immobilization of the molecule of interest. The desired region(or regions) of a surface coated with the photoinitiator is illuminatedto initiate the reaction only in that region. For example, ahydroxylated silicon substrate can be treated with a photoiniferter suchas N,N-(diethylamino)dithiocarbamoylbenzyl(trimethoxy)-silane (SBDC),which forms a self-assembled monolayer on the surface of the substrate.A methyl-methacrylate solution is then supplied, and UV irradiation ofthe desired region of the surface initiates polymerization to form asurface-tethered polymer brush of PMMA (e.g., including a couplinggroup) only in that region.

Another method of selectively immobilizing molecules of interest indesired regions on substrate surfaces involves the selective patterningof materials with different characteristics in different regions andrelying upon the differing characteristics of the surfaces in theselective immobilization process. In the exemplary ZMW substratesdescribed herein (as well as in other hybrid substrate types, e.g.,metal or semiconductor based sensors that rely on surface associatedmolecules of interest, e.g., ChemFETS), such patterned hybrid surfacesalready exist. In particular, ZMW substrates typically comprise a metalcladding layer, e.g., aluminum typically including an aluminum oxidesurface layer, deposited over a silica based layer, e.g., SiO₂, with anaperture disposed through the cladding layer atop the SiO₂ layer. Theresulting structure of the waveguides includes metal or metal oxidewalls, e.g., Al₂O₃ with a SiO₂ base. The aluminum oxide surface istypically relatively highly positively charged in aqueous solutionswhile the SiO₂ surface carries a substantial negative charge. Suchcharge differentials may be readily employed to selectively localize andimmobilize molecules of interest upon one surface relative to the other.

By way of example, DNA polymerase enzymes typically possess a relativelyhigh level of positively charged surface residues. As a result, apolymerase will generally be repelled by the positively charged metalcladding layer while being attracted and adsorbing to the negativelycharged glass surface at the base of a waveguide structure. Couplinggroups can be similarly deposited, and then polymerase (or anothermolecule of interest) coupled to the coupling groups. One may readilymodify the relative attraction/repulsion of the different surfaces byadjusting the nature of the environment to alter the charge of theenzyme, e.g., ionic strength, pH, additives, etc., by modifying eachsurface to enhance or reduce the charge component on the surface or byelectrically (dis)charging the metal, or by modifying the enzyme,coupling reagent, or other molecule of interest to adjust its level ofsurface charge, e.g., through mutation of the enzyme or through couplingto charged groups, e.g., polyions like polylysine, polyarginine, or thelike. In one aspect, after deposition of the polymerase (or other groupor molecule of interest) on the negatively charged surface, thepositively charged surface is passivated by coating it with an agentsuch as bovine serum albumin (e.g., acetylated BSA), polyglutamate, apolyelectrolyte, a polyelectrolyte multilayer, a polyelectrolyte-PEGcopolymer, a phosphonate, or a phosphate, as discussed in greater detailbelow. Such passivation can, for example, prevent nonspecific binding ofnucleotide analogs to the positively charged metal walls of a ZMW coreduring single molecule nucleic acid sequencing applications. In arelated aspect, passivation is accomplished prior to deposition of thepolymerase (or other group or molecule of interest), and optionallyfacilitates selective deposition, e.g., by blocking polymerase bindingto the walls.

As noted above, the surface charge of a material can, in someembodiments, be an active, tunable characteristic which can beaddressed, e.g., by pH tuning and/or by external polarization of thesurface. For example, tin oxide (a transparent material) can be doped tomake it conductive, and its surface charge (polarization) can bemodulated to a desired value.

Other surface selective chemistries may likewise be employed. Forexample, different phospholipid compositions have shown the ability, inthe presence and absence of calcium, to form different levels ofsupported phospholipid bilayers on metal oxide surfaces and silicondioxide based surfaces. By selecting the lipid composition and thepresence or absence of calcium, one can target deposition of molecules,either as blocking or coupling groups, onto the different surface types.For example, one can select a phospholipid that has high bindingselectivity for metal oxide surfaces and use it to block the metalportion of the surface. Alternatively, one can utilize a phospholipidwith an appropriate coupling group that has high binding selectivity forthe underlying glass substrate, and thus selectively couple additionalgroups to the transparent substrate. Examples of these selectivephospholipid compositions are described in, e.g., Rossetti, et al.,Langmuir. 2005; 21(14):6443-50, which is incorporated herein byreference in its entirety for all purposes. Briefly, phospholipidvesicles containing between 50% and 20% DOPS (dioleoyl phosphatidylserine) in DOPC (dioleoyl phosphatidyl choline), added to a hybridTiO₂/SiO₂ surface exhibit selective formation of the lipid bilayer onthe SiO₂ surface in the absence of calcium, whereas calcium presencepermits bilayer formation upon the TiO₂ surface as well.

As will be appreciated, one may employ the glass selective phospholipidbilayer (or other surface-selective composition) as the coupling groupsor may use it as a masking layer for a subsequent blocking layerdeposition upon the metallic layer. This would then be followed byremoval of the lipid bilayer from the glass substrate followed bycoupling of the molecules of interest.

Alternatively, physical/chemical differences between the differentsurfaces may be subjected to differential binding based uponspecifically selective chemistries. For example, specific groups thatassociate with particular metal groups may be employed to selectivelylocalize molecules to one surface relative to the other, e.g.,gold/thiol chemistries, etc.

As another example, silanes (e.g., methoxy-silane reagents) form stablebonds with silica surfaces via Si—O—Si bond formation, but do notsignificantly modify aluminum or aluminum oxide surfaces underappropriately selected reaction conditions (e.g., vapor phase favorsmodification of silica surfaces, as do certain conditions in solution).Silanes, for example, silanes modified with coupling groups forattachment of enzymes or other molecules of interest (e.g.,biotin-PEG-silanes such as those described in U.S. patent applicationSer. No. 11/240,662), can thus be used to selectively pattern hybridsubstrates such as ZMWs that contain silica surfaces. Ellipsometry andcontact angle measurements on Si surfaces previously modified with Al₂O₃show undetectable levels of silane reagent deposition. In addition,fluorescently labeled neutravidin does not bind specifically toAl₂O₃-modified fused silica slides after biotin-PEG-silane deposition onthe slides, while, in contrast, biotin-PEG-silane modification of fusedsilica slides (not modified with Al₂O₃) results in very high specificityof neutravidin binding via the biotin ligand. Such results demonstratethe feasibility of modifying only the fused silica bottom of a ZMW orsimilar device with little or no modification of the aluminum walls ortop surface of the device, using methoxysilane reagents.

As another example, negatively charged surfaces can be selectivelymodified by adsorption of copolymers containing positive polyelectrolyteblocks and PEG-ylated (or similar anti-fouling) blocks. The polycationicblocks bind to regions of the device that are electronegative, and thePEG components provide a nonreactive surface to preclude nonspecificbinding. Exemplary polyelectrolyte-PEG copolymers include PLL-PEG(poly(L-lysine)-poly(ethylene glycol)). The PEG groups, or a subsetthereof, can include a coupling group such as biotin or the other groupsdescribed herein (see, e.g., U.S. patent application publication2002/0128234 “Multifunctional Polymeric Surface Coatings in Analytic andSensor Devices” by Hubbell et al., Huang et al. (2002)“Biotin-Derivatized Poly(L-lysine))-g-Poly(ethylene glycol): A NovelPolymeric Interface for Bioaffinity Sensing” Langmuir 18(1): 220-230).Thus, for example, the SiO₂ surfaces of a ZMW can be coated withPLL-PEG-biotin, and biotinylated polymerase can then be coupled to thebottom of the ZMW via avidin or streptavidin binding to thePLL-PEG-biotin.

In one aspect, selective immobilization of the molecule of interest onone type of material in a hybrid substrate (e.g., a ZMW) is complementedor facilitated by modification of the other type of material. Forexample, for a ZMW that is to be used in an application such assingle-molecule nucleic acid sequencing, it is desirable to selectivelyimmobilize the polymerase to the bottom silica surface of the ZMW, andit is also desirable to passivate the metal walls and top surface of thedevice (before or after immobilization of the polymerase). Unmodifiedaluminum or aluminum oxide ZMW surfaces, which as noted above tend to bepositively charged in aqueous solution, can demonstrate undesirablenonspecific binding of proteins (such as neutravidin or streptavidin andpolymerase), nucleotide analogs (e.g., through the analog's phosphategroups), and dyes (e.g., dyes with sulfonic or carboxylic acid groups).As noted above, such undesirable electrostatic interactions can beminimized by binding of passivating agents to the surface; additionalexamples of suitable passivating agents include, but are not limited to,anionic polyelectrolytes such as poly(styrenesulfonate) and poly(acrylicacid) and macromolecules such as heparin and alginine.

In some instances, however, the adsorption of anionic polyelectrolytesto a positively charged surface may result in overcompensation of thenet charge of the surface, where adsorption of the polyanion results ina change in the net surface charge from positive to negative. Thischange in principle minimizes the nonspecific adsorption of nucleotideanalogs or other negatively charged compounds to the surface, but hasthe disadvantage that many proteins (e.g., polymerases) have affinityfor electronegative surfaces. Thus, an electronegative surface producedby such overcompensation may result in undesirably high levels ofpolymerase nonspecific binding. This problem can be addressed by usinghigh salt immobilization conditions; however, the high salt regime cancause swelling of the polyelectrolyte layer as well as partial loss ofpolyelectrolytes. In addition, coating of surfaces with polyelectrolytesis a dynamic process, and it is possible that the polymerase mayeventually form stable activity-blocking complexes with thepolyelectrolytes.

Optionally, instead of passivating the positively charged surface byadsorption of anionic polyelectrolytes, positively charged surfaces canbe passivated by binding of copolymer structures containingpolyelectrolyte blocks (negative) and PEG-ylated blocks. Thepolyelectrolyte blocks of the copolymer adsorb or anchor themacromolecules to regions of the device that are electropositive (e.g.,the aluminum or aluminum oxide areas of a ZMW), and the PEG componentsprovide a non-ionic cushion that precludes the surface attachment or thecomplexation of the polymerase with the polyelectrolyte blocks. Thepolyelectrolyte(PE)-PEG copolymers can, for example, be diblock (PEG-PE)or multi-block copolymers (e.g., PE-PEG-PE or PEG-PE-PEG), as well asbranched polymers, comb polymers, or dendron-like polymers. A fewexemplary linear and branched copolymers are schematically illustratedin FIG. 16 Panel VI. It will be evident that, while the exemplarycopolymers described herein employ PEG, any anti-fouling backbone isapplicable, for example, polypyrrolidone, polyvinyl alcohol, dextrans,and polyacrylamides. See, e.g., U.S. patent application publication2002/0128234, Voros et al. (2003) “Polymer Cushions to Analyze Genes andProteins” BioWorld 2:16-17, Huang et al. (2002) Langmuir 18(1): 220-230,and Zoulalian et al. (2006) J. Phys. Chem. B 110(51):25603-25605.

Orthogonal modification of a hybrid substrate with two compositions withdifferent selectivities for different surface characteristics isschematically illustrated in FIG. 16. As shown in Panel I, ZMW 1600includes core 1602 disposed through aluminum cladding layer 1604 totransparent silica substrate 1606. The aluminum core has a thin aluminumoxide layer 1605 on its surface. As shown in Panel II, the bottomsurface of the ZMW is selectively modified with a mixture ofbiotin-PEG-silane 1620 and PEG-silane 1622 (e.g., at a ratio selected toprovide a desired density of biotin coupling groups, and thus ultimatelyof molecules of interest, on the surface, optionally, one per core). Asillustrated in Panel III, the walls and top surfaces of the device arethen selectively modified with polyanion-PEG copolymer 1630. As shown inthe expanded view in Panel V, copolymer 1630 includes polyanion (A)blocks 1631 and PEG (B) blocks 1632. (It is worth noting thatmodification of the aluminum surface is optionally performed before,rather than after, modification of the silica surface.) Biotinylatedpolymerase 1608 is then bound via neutravidin 1609 to the biotincoupling group on biotin-PEG-silane 1620, as shown in Panel IV.

In one aspect, the compositions used to passivate the surface to whichthe molecule of interest is not attached (e.g., the aluminum surface)can also have a selected density of moieties that add functionality tothe surface. For example, in the PE-PEG copolymers described above,fluorescence quenching moieties 1640 can be attached to the functionalends of the PEG blocks (FIG. 16 Panel V). As another example, orthogonalligand schemes can be used to attach proteins to work in tandem withpolymerases or other molecules of interest; e.g., in embodiments inwhich biotin is used to immobilize polymerase 1608, functional group1640 can be a SNAP, HA, GST, or similar non-biotin coupling group, tobind a suitably modified second protein. These second proteins can beused to break up newly synthesized DNA chains, assist in removingreaction products from solution, assist in bringing reactants to theregion of reaction, assist in regeneration of triplet quenchers, or thelike.

As another example, the surface of the hybrid substrate to which themolecule of interest is not immobilized can be passivated using apolyelectrolyte multilayer. Polyelectrolyte multilayers are convenientlyformed through successive deposition of alternating layers ofpolyelectrolytes of opposite charge. See, e.g., Decher (1997) Science277:1232. Formation of a polyelectrolyte multilayer is schematicallyillustrated in FIG. 17. As shown in Panels I and II, in step 1positively charged substrate 1705 is contacted with polyanion 1732,which adsorbs to the surface of the substrate. Excess polyanion iswashed away in step 2. In step 3, a layer of polycation 1734 isdeposited over the layer of polyanion 1732 formed in step 1; excesspolycation is washed away in step 4. Steps 1-2 and/or 3-4 are repeatedas desired, to deposit alternating layers of oppositely chargedpolyelectrolytes and form multilayers of essentially any desiredthickness and resulting surface charge (negative when the polyanion isdeposited last, or positive when the polycation is deposited last).Panel III illustrates exemplary polycation poly(ethyleneimine) andexemplary polyanion poly(acrylic acid), which are optionally employed toform polyelectrolyte multilayers.

Optionally, the final layer in a polyelectrolyte multilayer comprises apolyelectrolyte-PEG copolymer, for example, a copolymer such as thosedescribed above containing polyelectrolyte blocks (either positive ornegative, depending on the charge of the preceding layer in themultilayer) and PEG-ylated blocks. As just one example, a poly(acrylicacid) layer in a polyelectrolyte multilayer can be followed by a layerof PLL-PEG or polyglutamate-PEG, to provide a PEG finish. It will beevident that, while the exemplary copolymers described herein employPEG, any anti-fouling backbone is applicable, for example,polypyrrolidone, polyvinyl alcohol, dextrans, and polyacrylamides.

Differential surface derivatization of a hybrid substrate with twocompositions having different selectivities for different surfacecharacteristics and formation of a polyelectrolyte multilayer isschematically illustrated in FIG. 18. As shown in Panel I, ZMW 1800includes core 1802 disposed through aluminum cladding layer 1804 totransparent fused silica layer 1806. The aluminum walls have a thinaluminum oxide layer 1805 on their surface. As shown in Panel II, thebottom surface of the ZMW is selectively modified with a mixture ofbiotin-PEG-silane 1820 and PEG-silane 1822. As illustrated in Panel III,polyelectrolyte multilayer 1830 is then deposited on the walls and topsurfaces of the device. The polyelectrolyte multilayer can be depositedas illustrated in FIG. 17, for example; a layer of polyanion (e.g.,poly(acrylic acid)) is deposited on the positively charged aluminumoxide layer 1805, followed by a layer of polycation (e.g.,poly(ethyleneimine)), then another layer of polyanion, etc. For singlemolecule sequencing or similar applications, the final layer of thepolyelectrolyte multilayer is typically a polyanionic layer, such thatthe surface of the polyelectrolyte multilayer is negatively charged torepel nucleotide analogs (or optionally a polyelectrolyte-PEG copolymeror similar, again to provide a surface with low binding to the analogs).As shown in panel IV, biotinylated polymerase 1808 is then bound vianeutravidin 1809 to the biotin coupling group on biotin-PEG-silane 1820.Such exploitation of the differences in surface properties of thematerials constituting a ZMW, e.g., silanization specific to the glassbottom and passivation of the sides and top aluminum oxide surfaces withpolyelectrolyte multilayers to prevent nonspecific binding, can limitpolymerase occupancy to the ZMW bottom, avoiding polymerase occupancy onside wall and top surfaces while limiting nonspecific binding ofnucleotide analogs or the like.

As yet another example of ways in which the different materials in ahybrid substrate can be differentially modified based on their differentsurface characteristics, phosphate and phosphonic acid compounds can beemployed (as can other compounds that exhibit surface specificchemisorption and/or self-assembled monolayer formation). Phosphate orphosphonic acid moieties bind strongly to metal oxides (e.g., aluminumoxide, titanium oxide, zirconium oxide, tantalum oxide, niobium oxide,iron oxide, and tin oxide) but do not bind strongly to silicon oxide.Thus, compounds that comprise at least one phosphate group (—OP(O)(OH)₂,whether protonated, partially or completely deprotonated, and/orpartially or completely neutralized) or phosphonic acid group(—P(O)(OH)₂, whether protonated, partially or completely deprotonated,and/or partially or completely neutralized) can be used to selectivelymodify the aluminum oxide surfaces of a ZMW or similar hybrid substrate.

For example, a metal oxide surface can be modified with an alkylphosphate or an alkyl phosphonate. (The terms phosphonic acid andphosphonate are used interchangeably herein.) Exemplary alkyl phosphatesand alkyl phosphonates include, but are not limited to, an alkylphosphate or alkyl phosphonate in which the alkyl group is a straightchain unsubstituted alkyl group (e.g., a straight chain alkyl grouphaving from 1 to 26 carbons, e.g., from 8 to 20 carbons, e.g., from 12to 18 carbons). Additional exemplary alkyl phosphates and alkylphosphonates include functionalized or substituted alkyl phosphonatesand alkyl phosphates, for example, functionalized X-alkyl-phosphonatesand X-alkyl-phosphates where X is a terminal group comprising orconsisting of a vinyl (CH₂), methyl (CH₃), amine (NH₂), alcohol (CH₂OH),epoxide, acrylate, methacrylate, thiol, carboxylate, active ester(NHS-ester), maleimide, halide, phosphonate, or phosphate group, or anethylene glycol (EG) oligomer (EG4, EG6, EG8) or polyethylene glycol(PEG), photo-initiator (e.g., photo-iniferters such as dithiocarbamates(DTC)), photocaged group, or photoreactive group (e.g., psoralen). Thealkyl chain spacer in the X-alkyl-phosphonate or X-alkyl-phosphatemolecule is a hydrophobic tether that optionally has 1 to 26 methylene(CH₂) repeat units, preferably from 8 to 20, and more preferably from 12to 18. The alkyl chain may contain one or more (up to all) fluorinatedgroups and/or can instead be a hydrocarbon chain with one or more doubleor triple bonds along the chain. The X-alkyl-phosphate orX-alkyl-phosphonate layer can furthermore be used as a substrate toanchor other ligands or components of the surface stack, such as apolyelectrolyte multilayer or chemisorbed multilayer. The alkylphosphates/phosphonates can form a stable, solvent resistantself-assembled monolayer that can protect the underlying material (e.g.,aluminum) from corrosion etc.; the role of the alkyl tether in the abovestructures is to enhance the lateral stability of the chemisorbedmonolayer in aqueous environments. In embodiments in which thephosphonate or phosphate compound includes an unsaturated hydrocarbonchain, the double or triple bond(s) can serve as lateral crosslinkingmoieties to stabilize a self-assembled monolayer comprising thecompound.

Specific exemplary alkyl phosphates and alkyl phosphonates include, butare not limited to, octyl phosphonic acid, decyl phosphonic acid,dodecyl phosphonic acid, hexadecyl phosphonic acid, octadecyl phosphonicacid, docosyl phosphonic acid (i.e., C22 phosphonic acid),hydroxy-dodecyl phosphonic acid (HO(CH₂)₁₂P(O)(OH)₂),hydroxy-undecenyl-phosphonic acid, decanediylbis(phosphonic acid),dodecylphosphate, and hydroxy-dodecylphosphate. Ellipsometry and/orcontact angle measurements show that octyl phosphonic acid, octadecylphosphonic acid, hydroxy-dodecyl phosphonic acid, and dodecyl phosphonicacid exhibit specificity toward aluminum/aluminum oxide surfacesrelative to Si/SiO₂ surfaces. Modification of metal oxides with suchphosphates and phosphonates has been described, e.g., in Langmuir (2001)17:3428, Chem. Mater. (2004) 16:5670; J. Phys. Chem. B (2005) 109:1441,Langmuir (2006) 22:6469, Langmuir (2006) 22:9254, Langmuir (2006)22:3988, J. Phys. Chem. B (2003) 107:11726, J. Phys. Chem. B (2003)107:5877, Langmuir (2001) 17:462, J. Phys. Chem. B (2006) 110:25603,Langmuir (2002) 18:3957, Langmuir (2002) 18:3537, and Langmuir (2001)17:4014.

Metal oxide surfaces can similarly be modified with polyphosphates orpolyphosphonates. Chemisorption, e.g., of polyphosphonates differs fromthe previous description of polyelectrolyte adsorption in that theligands (phosphonic acid moieties) form a chemical complex with thesubstrate (e.g., alumina, zirconia, or titania). Such interaction isstronger and less reversible to salt exchange than are simpleelectrostatic interactions. Examples include, but are not limited to,PEG-phosphonates such as those described in Zoulalian et al. (2006)“Functionalization of titanium oxide surfaces by means ofpoly(alkyl-phosphonates)” J. Phys. Chem. B 110(51):25603-25605 orPEG-polyvinyl(phosphonate) copolymers. (In general, copolymers includingchemisorbing moieties plus PEG or other anti-fouling moieties arecontemplated herein.)

Other suitable phosphonates include high molecular weight polymericphosphonates such as polyvinylphosphonic acid (PVPA)

end-capped phosphonates such as

(commercially available from Rhodia as Aquarite® EC4020 and Aquarite®ESL, respectively); andcopolymers such as vinyl phosphonic acid-acrylic acid copolymer

(commercially available from Rhodia as Albritect™ CP30).

Suitable phosphonates also include low molecular weight phosphonatessuch as 2-carboxyethyl phosphonic acid (also known as3-phosphonopropionic acid; commercially available from Rhodia asAlbritect™ PM2) and the compounds listed in Table 1 (commerciallyavailable as Dequest® compounds from Solutia, Inc., St. Louis Miss.).Phosphonate compounds can be supplied as salts (e.g., sodium, potassium,lithium, or ammonium salts) or, preferably, as free acids.

TABLE 1 Exemplary phosphonic acid compounds. Chemical Name StructureAmino tri (methylene phosphonic acid)

1-Hydroxyethylidene-1,1,- diphosphonic acid

Hexamethylenediaminetetra (methylenephosphonic acid)

Diethylenetriamine penta(methylene phosphonic acid)

ethylenediamine tetra(methylene phosphonic acid)

bis(hexamethylene triamine penta(methylenephosphonic acid)) Aminomethylene phosphonic acid 2-Phosphonobutane-1,2,4- tricarboxylic acid

Monoethanloamine diphosphonate

A few exemplary uses of phosphonates and phosphates follow, with respectto treatment of a ZMW where a molecule of interest such as a polymeraseis to be immobilized selectively on the bottom, silica surface of theZMW waveguide cores. It will be evident that similar considerationsapply to treatment of other hybrid substrates. As one example, a ZMWchip can be treated with a phosphonate to passivate the aluminum oxidesurface of the ZMW, and a positively charged polymerase can then beimmobilized by selective binding to the negatively charged silicasurface. Similarly, the ZMW chip can be treated with a phosphonate, acapture reagent that can be used for subsequent immobilization of thepolymerase (e.g., neutravidin) can be immobilized by binding to thesilica surface, and then the polymerase can be immobilized by binding tothe capture agent. In these examples, the phosphonate passivates thealuminum oxide surface, providing bias (e.g., by blocking the aluminumoxide) and providing a surface with low nonspecific binding ofnucleotide analogs, etc. In related examples, after immobilization ofthe polymerase or the capture agent, a polyelectrolyte multilayer isformed on the aluminum oxide surfaces to passivate them. Phosphonatesand phosphates can also be employed in combination with compounds thatselectively modify the silica surfaces of the ZMW. Thus, for example,the aluminum oxide surface can be passivated and/or blocked with aphosphonate, and silane reagent(s) can then be employed to modify thesilica surface (or vice versa, with modification of the silica surfacepreceding phosphonate deposition).

In one class of embodiments, the phosphate or phosphonate compoundserves as the first layer on which a polyelectrolyte multilayer is builton the surface, e.g., by successive deposition of oppositely chargedpolyelectrolytes as described above. In a related class of embodiments,a chemisorbed multilayer is formed on the surface. The chemisorbedmultilayer can include, e.g., alternating layers of amulti-phosphonate-containing reagent (for example, a diphosphonate, suchas 1,n-alkyl diphosphonic acid, or a polyphosphonate, such aspolyvinylphosphonate) and zirconium (IV) ligands. The zirconium (IV)ligand for the phosphonate can be provided by providing a precursor suchas zirconium t-butoxide, zirconium acetylacetonate, or zirconiumethoxide, from which the phosphonate displaces the ligand around thezirconium. The multilayers can be formed using methods well known in theart, for example, by alternately dipping the substrate or surface in asolution of the phosphonate and in a solution of the zirconium precursor(with an intermediate heat annealing step), or by alternating dipping ina solution of the phosphonate with organometallic chemical vapordeposition (MOCVD) or rapid thermal chemical vapor deposition (RT-CVD)of the zirconium (with an annealing step if necessary). Such chemisorbedmultilayers are robust, and are similar to polyelectrolyte multilayersbut with the advantage of having the equivalent of chemisorbed“cross-links” between adjacent layers rather than physical electrostaticones as in polyelectrolyte multilayers.

In another aspect, thermodynamic or diffusion limited processes may beemployed in the selective activation and/or deactivation of desired ornon-desired regions. In particular, active coupling groups may bedisposed over a substrate surface, including within ZMW structures, andmay be provided in active form. They are then subsequently andselectively deactivated by exposing the substrate to capping or blockinggroups that will prevent any additional coupling to those groups.Because the coupling groups that are present on the desired regionsreside within the ZMW, e.g., at the bottom surface, diffusion of thecapping or blocking agents to these groups is somewhat limited. As aresult, those coupling groups will be less prone to being blocked (willlikely be the last groups to be blocked), and may be used to couple themolecules of interest toward the bottom surface of the ZMW. Inparticular, by controlling the time of exposure of the substrate as awhole to the blocking agent, the concentration of the blocking agent,and other conditions of the capping reaction, e.g., temperature, etc.,one can control the degree to which the coupling groups within thewaveguide become blocked or capped. In this aspect of the invention, itwill be appreciated that the blocking component need not specificallybind to particular coupling groups to prevent coupling of the moleculeof interest. In some cases, such blocking or capping groups may preventsuch binding through its presence within the waveguide or other portionsof the surface. This may include hydrophobic or hydrophilic coatingmaterials that may form a thin or monolayer over the surface and thusblock binding of the molecule of interest, or which provide a spatial orsteric barrier to binding at a given coupling group without actuallybinding to the active coupling component of the coupling groups.

The foregoing aspects of the invention are schematically illustrated inFIG. 6. As shown, a waveguide structure 600 is provided with a uniformcoating of coupling groups 602 disposed upon it (shown as opendiamonds). Contacting the overall structure with capping groups 604(shown as closed circles) results in diffusion limited capping withinthe waveguide structure, and as a result, leaves more active (uncapped)coupling groups 602 toward the bottom surface of the waveguide structurefor coupling molecules of interest in a subsequent contacting step.

As will be appreciated, the initial step of providing active couplinggroups over an entire surface may be avoided where one simply wishes tocouple groups directly to the underlying surface, e.g., silanol groupson glass substrates, or the like. In particular, by initially blockingany active coupling groups on the surface for a relatively short period,those groups that are most accessible, e.g., not within the bottomregions of a ZMW, will be blocked first. A subsequent, longer exposureof the partially blocked or capped surface groups to coupling groupsthat are capable of binding to such surface groups will yield suchcoupling groups immobilized upon the bottom regions of the waveguidestructures. The amount of time, concentration, temperature, and otherconditions of each step may be varied to provide optimal conditions foreach of the blocking steps and coupling steps, and may be determinedbased upon readily identifiable characteristics and simpleexperimentation.

An alternative approach to additively providing molecules of interest ina desired location is through the optical trapping of the molecule inthe desired location, e.g., using optical “tweezer” techniques. Inparticular, using the strongly enhanced electric field created byfocused laser energy within an optical confinement, such as a ZMW, onemay enhance the concentration of particles such as molecules ofinterest, or enrich for their presence within the focal region of a ZMWand subsequently couple it to a binding group located within thatregion. The molecule of interest may be provided coupled to additionalgroups, e.g., avidin, streptavidin, neutravidin, biotin, or particles,such as beads, e.g., heparin sparse beads, or the like, etc., in orderto provide a sufficiently large particle for trapping. The use of suchoptical trapping/enhancement techniques has been described in detail forexerting trapping forces on particles as small as 10 nm. See, e.g.,Novotny, et al., Phys. Rev. Letts. 79(4):645-648 (July 1997), which isincorporated herein by reference in its entirety for all purposes.

As an alternative or additional process to the selectiveactivation/deactivation processes discussed above, or below, the presentinvention optionally or additionally may include an initial patterningstep to provide neutral or inert groups upon areas where it is notdesired to couple the molecules of interest. Such patterning typicallyprovides a coarse selectivity to the localization, in that it is notspecifically intended to yield the final selective surface. For example,in the context of micro or nanowells, or other structures provided in anotherwise planar surface, inert groups may be printed, applied orotherwise patterned upon the upper planar surface of the substratewithout depositing such materials into the nanostructures, e.g., ZMWs.By first blocking the non-relevant surfaces with inert groups, one canthen deposit and couple active groups within the relevant areas. Again,in the context of a ZMW array, depending upon the density of the array,e.g., the percentage of overall substrate occupied by waveguidestructures, a substantial amount of non-relevant surface can be blockedand thus prevented from harboring molecules of interest that mightotherwise interfere with the ultimate application of the device, e.g.,through substrate depletion, excessive product formation, etc.

Such patterning may include simple stamping of the inert molecules ontoa surface whereby the inert groups will not penetrate the depressions onthat surface, or it may involve more complex printing patterns usingeither nanolithographically produced stamps to provide selectivedeposition, ink jet printing, or the like, to selectively deposit inertgroups upon the overall substrate surface. An example of the process ofthe invention is schematically illustrated in FIG. 7.

As shown, a substrate 700 that includes an array of ZMWs 702 disposed inits surface 704 (in panel I), is contacted with a separate substrate 706bearing a printable material 708 thereon that prevents coupling ofactive functional groups to the substrate surface 704 (Panel II). Bycontacting surface 704 with the printable material 708, the material istransferred to the surface 704 while not penetrating ZMWs 702 (PanelsIII and IV). As a result, subsequent coupling of molecules of interestto the upper surface 704 of substrate 700 is blocked. The printablematerial may include any of a variety of different materials, including,e.g., inert surface associating groups that simply cap any active groupson the surface. Alternatively, such material may include coatingmaterials that prevent association with the molecules of interest, e.g.,hydrophobic or hydrophilic materials, highly charged materials thatrepel any binding or other association, or materials that provide animpenetrable barrier to such materials, e.g., polymer coatings, resists,or the like.

As will be appreciated, any of the foregoing processes may be practicedin conjunction with other processes described herein to further enhancesurface selectivity and/or localization.

III. SUBTRACTIVE PROCESSES

As noted previously, in alternative aspects, subtractive processes areemployed to provide the molecule(s) of interest in the desired regionsof a substrate and at the desired concentration and/or density ofmolecules. As noted, subtractive processes are generally characterizedand differentiated from the additive processes described above, in thatthey deposit the molecule of interest more ubiquitously, e.g., over anentire substrate surface including in the desired regions. Subsequently,the excess molecules of interest, e.g., that are located in non-desiredregions, are removed. A variety of different processes may be employedin such subtractive processes.

In one example, a process may be employed that is roughly the inverse ofthe photoactivatable processes described above. In particular, couplingof the molecule of interest may be accomplished over the substratesurface using a selectively cleavable linker or coupling group. Avariety of photocleavable linker chemistries are known in the art andinclude 2-nitrobenzyl linkers (See, e.g., Rodebaugh, R.; Fraser-Reid,B.; Geysen, H. M. Tetrahedron Lett. 1997, 38, 7653-7656), as well as anumber of other known photocleavable linker types, see e.g., Org. Lett.,2 (15), 2315 -2317, 2000.

In the context of the present invention, a coupling group may be broadlyapplied to a substrate surface using a photocleavable linker group. Themolecule of interest is then coupled to the coupling groupssubstantially non-selectively. Selective illumination of areas that areoutside the desired regions then releases the molecules of interest fromthese areas, leaving such molecules substantially only coupled withinthe desired regions. Washing of the substrate then removes the moleculesfrom any potential interference with the desired application.

This aspect of the invention is schematically illustrated in FIG. 8. Inparticular, coupling groups 802 (shown as open diamonds) are provided ina uniform coating over the surface of the waveguide structure, but areattached to that surface through photocleavable linker groups 804 (shownas filled circles). The surface that is outside of the area of interest,e.g., not at the bottom surface 806 of ZMW core, is then exposed tolight (shown as wavy arrows 808) to cleave the linker groups in thenon-desired regions, where coupling is not ultimately desired, leavingthose coupling groups in the desired regions for subsequent coupling,e.g., at bottom surface 806, available for coupling.

Another subtractive approach to the selective immobilization ofmolecules of interest, particularly within nanostructured wells or otherconstrained spaces, e.g., optical confinements like ZMWs, utilizesdeactivation components, e.g., that deactivate either the molecule ofinterest or the component linking that molecule to the surface, orotherwise cause the digestion, deactivation, release or removal of suchmolecules from the surface. For ease of discussion, such components arereferred to herein as “deactivation components” regardless of whethersuch components degrade and/or digest the molecules of interest,inactivate such molecules, e.g., through nonreversible binding to activesites or other modification of such molecules of interest, or the like,or merely release them from the surface, e.g., through the cleavage of alinking group or otherwise.

Such approaches may rely upon thermodynamics to selectively avoiddeactivation or removal within a ZMW, as diffusion of largerdeactivation components, e.g., enzymes, i.e., proteases or other largermacromolecular compounds, or the like, will diffuse into a waveguidemore slowly, similar to the diffusion limited capping of coupling groupsshown in FIG. 6.

Alternatively, the method may rely upon the use of additional componentsto prevent the deactivation components from accessing the molecules ofinterest within the constrained space, e.g., a ZMW. One particularlypreferred aspect of such prevention involves the coupling of thedeactivation component to a large component, such as a bead or otherparticle, or a large polymeric molecule or aggregation of molecules,that are at least partially incapable of entering into the ZMW. Suchlarger components are generally referred to as exclusionary componentsas they are sized or shaped to be at least partially excluded fromrecesses such as ZMWs on substrates. Because the deactivation componentis coupled to the exclusionary component, it is only capable or morecapable of accessing molecules of interest that are exposed upon orproximal to the upper surface of the substrate incorporating the ZMW(s),and are thus accessible to the deactivation component, and not thosemolecules that are well within the structures.

In accordance with this aspect of the invention, the deactivationcomponent might include digestive molecules, e.g., proteases, such asserine proteases, i.e. proteinase K, subtilisin, and the like, threonineproteases, aspartic acid proteases, cysteine proteases,metalloproteases, and glutamic acid proteases, e.g., for digestion,cleaving or release of protein or peptide based molecules of interest orlinking components in either non-specific or specific fashion, e.g.,using a target protease to cleave a particular linking molecule, e.g., abiotin. Alternatively, such deactivation components might includecarbohydrate digesting enzymes (also termed carbohydrases), such ascellulases and amylases, or nucleases, such as exo- or endonucleases,etc., for the digestion or cleaving of carbohydrate or nucleic acidbased linking molecules or the molecules of interest. This aspect of theinvention is schematically illustrated in FIG. 9.

As shown, an array 900 of confining structures, e.g., ZMWs 902, isprovided with molecules of interest 904 randomly deposited over itsentire surface, e.g., including the surface of cladding layer 908 andsubstrate layer 915 (Step I). Large particles, such as beads 912, havingdeactivation components immobilized upon their surface (or componentsthat otherwise deactivate, cleave or release the molecules of interest),are then contacted with the array 900. Because beads 912 are larger thanthe openings to the waveguides 902, the deactivation componentsimmobilized on the beads are only capable of accessing and inactivating,digesting, cleaving or releasing molecules of interest that aredeposited on surfaces outside the structures 902 or that aresufficiently proximal to the opening of such structures as to beaccessible by the immobilized components on the beads 912. As a result,molecules upon or near the surface outside of the ZMW structures areremoved or otherwise deactivated, leaving only those molecules that arewell within the constrained or exclusionary space of the waveguide (StepIII). This aspect of the invention is also further illustrated, below.

In related aspects, the beads may be provided with a binding orcrosslinking component that binds or crosslinks with or to the moleculeof interest. Subsequently, the bead may be mechanically removed from thesurface taking at least a portion of the molecules of interest with it.

A variety of different types of beads may be used, including beadsgenerally used in chemical and biochemical analyses, i.e., agarose,acrylic, silica, or polyacrylamide beads or the like, or otherchromatographic or enzyme immobilization media/matrices, such as F7m orG3m matrices, available from MoBiTec, GmbH (Gottingen, Germany),magnetic beads or other metallic beads. Similarly, methods for linkingthe deactivation component to the beads may be varied to achieve desiredresults. For example, linker groups having varied lengths may be used topermit penetration of the deactivation component partially into a ZMW orother constrained space. Likewise, linker stiffness may be adjustedthrough the chemical structure and/or crosslinking of the linkers toprovide greater or lesser ability for the deactivation component toenter into a confined space such as a ZMW.

In an alternative approach to the use of beads, other scaffold materialsmay be used to support the deactivation component and provide thatcomponent with accessibility to the upper surface of the overallsubstrate, and in some cases, a subset of the surfaces within recesseson that surface, e.g., a waveguide core. In particular, the scaffoldcomponent would result in the deactivation component being not entirelyexcluded from a given recess on the substrate surface, e.g., a zero modewaveguide core. By way of example, the deactivation component may beprovided tethered or coupled to a scaffold or supporting molecule thatis either only partially excluded from the recess or is only excludedwhen provided in certain orientations. For example, a rigid orsemi-rigid linear molecule, such as a double stranded nucleic acid orother rigid or semi-rigid elongated polymer, may be used that includesthe deactivation component, e.g., a protease, coupled to it at anintermediate position. The supporting molecule is provided of sufficientlength that it can only move into the recess if oriented appropriately,e.g., lengthwise. As a result of entering the recess lengthwise or beingretained upon the upper surface, only those molecules on the uppersurface or within the recess but within reach of the deactivationcomponent will be deactivated. By way of analogy, the supportingmolecule and intermediate deactivation component function as a chimneysweep to remove molecules of interest from the upper surface of thesubstrate and a certain distance within the recesses, as dictated, atleast in part, by the intermediate positioning of the deactivationcomponent on the supporting molecule.

In the case of a relatively typical zero mode waveguide structure ofapproximately 100 nm in depth and 70 nm in diameter, for example, adouble-stranded DNA oligonucleotide 150 nm in length could be used withthe deactivation component, e.g., a protease or the like, affixed to it.Positioning and coupling are accomplished through covalent couplingchemistry to a nucleotide analog that has been inserted in theoligonucleotide sequence at a selected position a given distance fromone or both ends. Because double-stranded DNA is mechanically rigid, thecenter portion of the oligonucleotide to which the deactivationcomponent is affixed is away from the end of the supporting molecule.Upon entry into a waveguide core, only the end of the supporting doublestranded DNA molecule will be able to reach the bottom of the core, andthus the deactivation component will be geometrically constrained awayfrom the bottom of the core, or other confined space. Thus, molecules ofinterest that are on the top surface or on the side walls of (forexample) a ZMW would be removed, while a molecule of interest on or nearthe floor of the ZMW, e.g., within the illumination volume, wouldremain. Positional coupling of deactivation components to doublestranded nucleic acids may be carried out by a variety of methods. Forexample, in the case of coupling proteins, such as proteases or otherenzymes, to nucleic acid supporting molecules, a protease or otherenzyme can be maleimide activated by conjugation with a bifunctionalcrosslinker such as GMBS (available from PIERCE). Thismaleimide-activated protein can be directly coupled to a single strandor double strand of DNA possessing an internal thiol modification (e.g.,a THSS internally labeled molecule available from, e.g., Operon, Inc.).The thiol modification is capped via a disulfide which is removed duringthe conjugation by TCEP (also available from PIERCE). Similarly, anucleic acid with an internal thiol can be conjugated with aheterobifunctional crosslinker (e.g., MAL-NHS,maleimide-N-hydroxysuccinimide) and then conjugated to a protease via anamine-NHS reaction. Similar reactions can be employed to conjugateamino-modified DNA to a protease with thiols available on or near itssurface.

The foregoing process is schematically illustrated in FIG. 13. As shown,a ZMW device 1300 includes a core 1302 disposed within a cladding layer1304, again extending to an underlying transparent substrate 1306. Asshown in panel I, a number of active molecules of interest, e.g.,polymerase molecules 1308, are adsorbed or otherwise coupled to thesurface of the overall substrate, including both within a desiredillumination region (as indicated by dashed line 1310), on upper surface1312 and at the upper wall surfaces of the core 1302. In the context ofthe invention, and as shown in panel II, a deactivation component, suchas protease molecule 1314, is coupled at an intermediate position to arigid, linear or elongated supporting molecule, such as dsDNA molecule1316. Because of its size and structural rigidity, the supportingmolecule 1316 with associated deactivation component 1314 onlypenetrates the core 1302 of the waveguide structure 1300 in an end-onorientation, or it lays across the upper surface 1312 of the overallstructure. As a result of this, only polymerases that are disposed uponthe upper surface or within reach of the deactivation component thatpenetrates a partial distance into the waveguide core will bepotentially affected by the deactivation component. As such, polymerasemolecules that are disposed at or near the bottom surface of thewaveguide core, e.g., within the illumination region, will be spareddeactivation (Panel III). As will be appreciated, the positioning of thedeactivation component and/or the rigidity of the supporting moleculemay generally be chosen to adjust the depth within a core structure atwhich deactivation can occur.

As noted above, the deactivation component is optionally a protease suchas Proteinase K that nonspecifically digests the active molecule or acoupling group etc., thereby removing it from the surface of thesubstrate. In other embodiments, the deactivation component is asite-specific protease (e.g., enterokinase, thrombin, TEV protease, orany of the variety of other site-specific proteases available in theart). Use of a site-specific protease can avoid autoproteolytic cleavageof the protease from the exclusionary component, which would releasesoluble active protease able to undesirably access the optimal confinedillumination volume of the structures.

An exemplary embodiment employing a site-specific protease isschematically illustrated in FIGS. 14A-14B. As shown, a ZMW device 1400includes a core 1402 disposed within a cladding layer 1404, againextending to an underlying transparent layer 1406. In this example,polymerase molecule 1408 is covalently linked to biotin 1420 throughpeptide linker 1421, which includes a cleavage recognition site forsite-specific protease 1415. Binding of biotin 1420 to streptavidin1409, which is in turn bound to biotin 1422 that is adsorbed orotherwise coupled to the surface of the substrate, couples polymerase1408 to the surface. As shown in FIG. 14A, a number of active moleculesof interest, e.g., polymerase molecules 1408, are coupled to the surfaceof the overall substrate, including both within a desired illuminationregion (as indicated by dashed line 1410) and at the upper wall surfacesof the core 1402 (and optionally also on upper surface 1412). As shownin FIG. 14B, cleavage of linker 1421 by protease 1415 releasespolymerase 1408 from the surface. The site-specific protease molecule1415 is coupled at an intermediate position to a rigid, linear orelongated supporting molecule, such as dsDNA molecule 1416. As for theembodiments described above, because of its size and structuralrigidity, the exclusionary component 1416 with associated protease 1415only penetrates the core 1402 of the waveguide structure 1400 in anend-on orientation, or it lies across the upper surface 1412 of theoverall structure. As a result of this, only polymerases that aredisposed upon the upper surface or within reach of the deactivationcomponent that penetrates a partial distance into the waveguide core arepotentially affected by the deactivation component. As such, polymerasemolecules that are disposed at or near the bottom surface of thewaveguide core, e.g., within the illumination region, will remainattached to the surface since their linkers are inaccessible to theprotease and are not cleaved.

Another exemplary embodiment employing a site-specific protease isschematically illustrated in FIGS. 15A-15C. As shown, ZMW device 1500includes core 1502 disposed within cladding layer 1504 that extends tounderlying transparent layer 1506. In this example, as illustrated inFIG. 15A, biotin coupling group 1522 is coupled to the surface of theoverall substrate via peptide linker 1521, which includes a cleavagerecognition site for site-specific protease 1515. Cleavage of linker1521 by protease 1515 releases biotin 1522 from the surface. Sinceprotease 1515 is coupled to exclusionary component double-stranded DNA1516, as shown in FIG. 15B the protease removes biotin 1522 from thesurface everywhere except the lowest portion of core 1502. As shown inFIG. 15C, streptavidin 1509 (or neutravidin etc.) and polymerase 1508coupled to biotin 1520 are then deposited on the substrate and areretained by binding to biotin 1522 only in optimal confined illuminationvolume 1510.

Another alternative subtractive method for the selective localization ofmolecules of interest involves the use of that molecule's own activityagainst it within the undesired regions. For example, in the case ofimmobilized nucleic acid polymerase enzymes, it has been determined thatsuch enzymes, when incorporating fluorescently labeled nucleotides underexcitation illumination, can suffer from substantial inactivation as aresult of photodamage. In accordance with the subtractive aspects of thepresent invention, by subjecting enzymes at the upper surface of awaveguide substrate to prolonged illumination during nucleic acidsynthesis in the presence of fluorescently labeled nucleotides ornucleotide analogs, one can effectively inactivate those molecules. Aswith the activation/inactivation based additive approaches describedabove, it will be appreciated that damaging illumination would notpenetrate to the bottom surface, or area of interest, of the ZMW, andthus, such enzymes present at these locations would remain active.Fluorophore mediated inactivation of polymerases is discussed at lengthin commonly assigned U.S. patent application Ser. No. 11/293,040, filedDec. 2, 2005, and incorporated herein in its entirety for all purposes.Other enzyme/fluorescent substrate pairs would be expected to yieldsimilar characteristics, e.g., ATP binding proteins/fluorescentlylabeled ATP. Additionally, other components may be employed thatgenerate radicals upon irradiation, that will damage those moleculesthat are within diffusive contact. By illuminating the upper surface ofa waveguide structure in the presence of such compounds, for example,one could generate oxygen or other free radicals, that will deactivatemolecules of interest within diffusive reach of such compounds. Avariety of such compounds are known in the art and include, e.g.,methylene blue, hypocrellin A, hypocrellin B, hypericin, Rose BengalDiacetate, Merocyanine 540, and other dyes available from, e.g.,Invitrogen/Molecular Probes (Eugene, Oreg.).

In another aspect of the invention, the structural characteristics of asubstrate may be actively employed in subtractively selecting moleculesof interest. In particular, substrates including optical confinements,such as ZMWs, typically include a metal layer deposited upon atransparent layer, e.g., glass or quartz, through which the waveguidesare disposed, exposing the transparent substrate at the bottom surfaceof the waveguide. In accordance with the invention, an overall substratethat includes molecules of interest both coupled to the metal layer andthe glass layer may be selectively partitioned, e.g., removing moleculesof interest from the metal surfaces, by applying an electrical potentialbetween the metal layer and the solution deposited over it, e.g.,through the use of an electrode in contact with such fluid. Because theunderlying substrate is not electrically conductive, the field betweenthe surface of the substrate and the fluid will be substantially lessthan that between the metal layer and the fluid. The electricalpotential may then be employed to selectively drive the molecules ofinterest from the metal surface and into solution (see FIG. 10). Thisdriving force may be selected and/or controlled to result inelectrophoretic forces, e.g., driving charged molecules of interest awayfrom the surface in the non-desired surface regions or driving cappinggroups toward such surfaces, or alternatively or additionally, changesin the local environment at the metal surface, e.g., pH changesresulting from the generation of protons at the metal surface, thatresult in release from the surface, e.g., through the use of acid labilelinkers, charge based linkages, e.g., hydrogen bonding, hydrolyticdegradation of molecules of interest on the metal surfaces through thegeneration of locally harsh environments, or the like.

In another aspect, electrochemically releasable linker compounds may beemployed to release molecules of interest from electrically activesurfaces. By way of example, linking molecules that includeelectrochemically controllable coupling may be patterned upon theoverall surface of a hybrid (metal/insulator) substrate. Applying acurrent through the metal portion of the surface results in release ofthe coupled molecule. Examples of such electrically switchable linkersinclude self assembled monolayers of biotin linked to quinone propionicester bearing linker compounds, i.e., alkanethiolates on gold surfaces.Application of a potential to the underlying metal substrate results inreduction of the quinine to hydroquinone that rapidly undergoeslactonization with the release of the tethered molecule, e.g., biotin(See, e.g., Hodneland, et al., J. Am. Chem. Soc. 2000, 122:4235-4236).

In addition to the use of such methods in optical confinements, it willbe appreciated that such electrophoretic and/or electrochemicalselection and immobilization processes may be similarly applied to otherhybrid analytical substrate types, including, e.g., metal orsemiconductor based sensors that rely on surface associated molecules ofinterest, e.g., ChemFETS (chemical field effect transistors), and thelike. In particular, the metal or semiconductor sensor element may beemployed as one electrode in the repulsion or attraction of differentgroups from or to the surface of the sensor to enhance coupling.

Other subtractive processes may employ lift-off methods where anotherwise active surface is coated with a lift-off layer that entrainsthe molecules of interest on the upper surface of the substrate, and insome cases penetrating a certain distance into a ZMW. Lifting off of thelayer brings the entrained molecules of interest with it, allowing thosenot entrained, e.g., those at the bottom surface of the ZMW, to remain.

This technique is schematically illustrated in FIG. 11. As shown, auniform or random distribution of molecules of interest 1104 isdeposited over a substrate 1100 that includes selected regions wheresuch molecules are desired (Step I). In the case of FIG. 11, such areasinclude optical confinements like ZMWs 1102. A coating layer 1106 isthen deposited over the surface as a viscous liquid, e.g., having aviscosity of 1 or greater (Step II). Because of its relative viscosityand the relatively small dimensions of the waveguides 1102, and/or thematerial's relatively slow diffusion in a liquid material present in thewaveguide core, the coating layer 1106 will typically not flowcompletely into the waveguide structure. The coating layer is thentypically allowed to cure, e.g., through air drying, heating or exposureto UV radiation, chemical crosslinking, entraining molecules of interestwithin the coating layer, e.g., molecules of interest 1108. Uponremoval, any molecules of interest entrained in the coating layer areremoved as well, leaving only those molecules of interest that were wellwithin the waveguide structure, e.g., molecules 1110 (Step III).Although the above described method relies upon the limited ability ofthe coating layer to penetrate the waveguide structure to leavemolecules of interest within such structures, it will be appreciatedthat such methods may be applied in the absence of such constrainedstructures. For example, the coating layer may be selectively patternedupon the surface, e.g., through screening or ink jet printing methods,to entrain and remove molecules of interest from selected regions.

Another subtractive, selective immobilization process relies generallyupon masking strategies to ensure localization of the molecule ofinterest where desired. In particular, such masking strategies typicallyutilize a masking layer that may be either removed to eliminatemolecules of interest from undesired locations, or which is depositedover a uniformly distributed population of the molecules of interest torender those in undesired locations inaccessible to the desiredoperation.

Other simpler brute force techniques are also within certain aspects ofthe invention, particularly related to subtractive processes. Forexample, one may use simple ablative processes to remove coupling groupsfrom exposed surfaces, e.g., surfaces upon or near the upper surfaces ofwaveguide array substrates. Removal of such groups would be expected toreduce the amount of molecules of interest that are bound to surfacesoutside of the waveguide structure. Such ablative processes include,e.g., laser ablation techniques, high sheer fluid ablation techniques,mechanical abrasion techniques, and the like that will remove materialsupon contact or exposure. By directing such ablative processes at theupper surfaces, it is expected that little or none of the ablativeforces will propagate into waveguide structures. Additional adjustmentsmay be made to further enhance the selectivity of the process. Forexample, using laser ablation techniques, one could direct the beam atan oblique angle to the upper surface of the substrate, therebypenetrating only a minimal distance into high aspect ratio recesses,e.g., ZMWs. Likewise, ablation energy could be modulated to focus onregions that did not include the regions where eventual coupling ofmolecules of interest is desired, e.g., focused upon substrate surfaceregions or spaces between ZMWs in an array.

Once the coupling groups have been provided upon the surface of thesubstrate, e.g., in the desired regions such as at the bottom surface ofa ZMW, the molecules of interest are then coupled to those activegroups. As noted elsewhere herein, coupling may be via functionalchemical groups, e.g., hydroxyl groups, amino groups, epoxy groups orthe like. Alternatively, coupling may occur through specific bindingpartners, e.g., where one member of a specific binding pair is thecoupling group attached to the surface (or is attached to a couplinggroup that is attached to the surface), and the other member of thebinding pair is attached to or is integral with the molecule ofinterest. In particularly preferred aspects, such specific binding pairsare used to couple the molecule of interest to the surface, including,e.g., the use of avidin, streptavidin or neutravidin as one member ofthe binding pair, and biotin as the other member. Additionally, sandwichbinding strategies may be employed, e.g., coupling biotin to the surfacein the area of interest, followed by linkage to avidin, which is inturn, linked to a biotin molecule coupled to the molecule of interest.Typically, a linker silane group is used as the initial functionalgroup. This group may be provided directly upon the surface or, asalluded to previously, diluted with similar linker silanes that areinert to additional coupling. In particularly preferred aspects, alinker silane bearing, e.g., a biotin group is immobilized in theinitial step, followed by coupling of a molecule of interest, e g., apolymerase enzyme, through a bridging avidin group coupled with anenzyme linked biotin group. As will be appreciated any of a variety ofdifferent configurations may be practiced within the context of theinvention.

In the case of molecules of interest that are enzymes or otherwiseactive proteins, the orientation of immobilization may be an importantcharacteristic to optimizing activity of the enzyme. For example, in thecase of DNA polymerases, random adsorption of polymerases to a surfacecan yield substantially less than 100% activity at least partially as aresult of some molecules being oriented in a way so as to prevent themfrom exhibiting optimal activity. As such, it may be desirable toprovide for a specific orientation of the molecule by providing ananchoring group or groups on the molecule to increase the probability ofcorrect orientation. Such methods have been previously described incommonly owned U.S. patent application Ser. No. 60/753,446, filed Dec.22, 2005, and incorporated herein by reference in its entirety for allpurposes. Alternatively, one may provide the enzyme with a substratemolecule or substrate proxy that can prevent surface adsorption in amanner that blocks the active site of the enzyme. By way of example, ithas been determined that immobilization of nucleic acid polymeraseenzymes, such as DNA polymerases, in the presence of template nucleicacid molecules yields substantially higher activity of surfaceimmobilized polymerases. Without being bound to a particular theory ofoperation, it is believed that the presence of the template moleculewithin the active site of the polymerase prevents immobilization of thepolymerase in a manner that interferes with the active site, due tosteric or other interference from the associated template. Whiletemplate nucleic acid molecules can be used, other template-likemolecules may also be used, including, e.g., LNA polymer strands, PNApolymers, or other nucleic acid analogs.

IV. EXAMPLES Example 1: Photoactivatable Groups for SelectiveImmobilization of DNA Polymerases

A substrate may be used that includes a glass substrate layer with analuminum cladding layer deposited over the glass layer. An array of ZMWcores is fabricated into the cladding layer to provide apertures throughthe cladding layer to the glass substrate. The overall substrate isoptionally further treated to provide a thin insulating layer over thecladding layer and cores, e.g., to provide a substantially uniformsurface. Such layers typically include SiO₂ coatings applied by vapordeposition techniques, including, e.g., CVD and MVD methods, as well asother methods such as fluid deposition or in situ formation using, e.g.,spin on glass systems. The substrate surface is derivatized to firstprovide a relatively uniform population of amino terminated groupscoupled to the surface. For example, for glass surfaces, suchderivatization typically employs standard aminosilane chemistries knownin the art. Alternatively, amine groups may be provided upon a linkermolecule that is coupled to the surface through existing hydroxyl groupsor surfaces otherwise derivatized. Such coupling groups may be providedat limited densities in order to further control the density of themolecules of interest that will ultimately be bound to the surface (see,e.g., commonly assigned U.S. patent application Ser. No. 11/240,662,filed Sep. 30, 2005, incorporated herein by reference in its entiretyfor all purposes).

Biotin molecules capped with an appropriate photolabile protectinggroup, such as MeNPOC, are then coupled to the derivatized surface usingknown chemistries, e.g., through an included epoxy group on the biotinmolecule.

Following washing of the surface, appropriate illumination radiation isdirected at the substrate through the transparent glass substrate layer,illuminating and deprotecting only the biotin groups at or near thebottom surface of the ZMW. DNA polymerase enzyme linked to avidin,streptavidin or neutravidin is then contacted with the substrate andselectively binds with the exposed biotin at the bottom of thewaveguides.

In a second exemplary process, a photoactivatable acid group, e.g.,surface coupled α-methylphenacyl ester, is coupled to the surface in thesame fashion provided above. Illumination, e.g., at 313 nm, through theZMW yields the acid groups at the bottom surface of the waveguides,which are then contacted with amino biotin groups followed by couplingto avidin linked polymerase enzymes, to yield enzyme groups only at ornear the bottom surface of the waveguide.

Example 2: Selective Digestion of DNA Polymerase Enzymes Using BeadBound Proteases

ZMWs that had previously been plasma treated in the presence of a PDMSgasket (to provide a priming layer), were provided with Φ29^(N62D) DNAPolymerase (complexed with a circular template nucleic acid)substantially uniformly surface adsorbed over the entire surface of thearray, including upon the upper surface of the cladding layer.

The array was then contacted with beads bearing immobilized Proteinase-K(Sigma Chemical Co., P0803 or P9290) for 5 minutes at room temperaturein 25 mM Tris-HCl, pH 7.5, 10 mM β-mercaptoethanol, 1 mM EDTA. The beaddiameter far exceeded the nominal diameter of the waveguide cores on thearray, preventing entry to the bead or its associated protease moleculesinto the cores to any substantial degree.

Polymerization reaction mixture including four dNTPs was then contactedwith the array under conditions suitable for DNA synthesis (50 mMTris-HCl, pH 7.5, 75 mM KCl, 20 mM (NH₄)₂SO₄, 10 mM β-mercaptoethanol,0.7 mM MnCl₂), and synthesis was allowed to proceed for 30 minutes at30° C.

Following synthesis any synthesized DNA on the array was stained withSybrGold stain. The array was then imaged using a standard fluorescencemicroscope. The array images, as well as images of the negative controlexperiment, are shown in FIG. 12. As shown in the negative control (RowI), bottom side illumination (Column A) shows the presence of asignificant amount of DNA within the waveguide structures, while topside illumination and observation (Column B) shows a uniform layer ofDNA produced over the entire surface of the array. In the proteinasetreated array (Row II), both the bottom side (Column A) and top side(Column B) show a similar pattern of DNA presence within specificwaveguides. Further, as can be seen, there is little DNA present uponthe upper surface other than within waveguides in the array, showing asubstantial reduction from the high level of DNA synthesis present inthe control experiment. Also of note is that the waveguides showing DNApresence from the upper surface track to the same waveguides showing DNApresence from the lower surface, indicating that DNA synthesis isoccurring within the waveguide structure, and not outside the waveguidecore. This also indicates that DNA being synthesized within thewaveguide structure is of substantial length, e.g., greater than 500bases, potentially up to 1000 or more bases in length, as it spans theillumination regions at the top and bottom portions of a waveguidestructure having a core region of approximately 70 nm in diameter and100 nm deep.

DNA synthesis experiments were also carried out in the presence oflabeled nucleoside polyphosphate analogs, labeled at the terminalphosphate group (see, e.g., Published U.S. Patent Application No.2003-0044781 and Levene, et al., Science (2003) 299:609-764, the fulldisclosures of which are incorporated herein in their entirety for allpurposes). These assays indicated substantially better signal to noiseratios than waveguide arrays that were not proteinase treated, showingmarkedly less interference from other noise sources, e.g., labeled byproducts of the polymerase reaction. As a result, it appears clear thatprovision of molecules of interest such as polymerase enzymes onlywithin a desired region of an analytical substrate, i.e., an observationregion, can have profoundly beneficial results on the application towhich the substrate is to be put.

Example 3: Selective Immobilization of DNA Polymerases by DifferentialModification of Surfaces

The following sets forth a series of experiments that demonstrateselective immobilization of a DNA polymerase on the bottom surface ofZMWs and passivation of the remaining ZMW surfaces with apolyelectrolyte multilayer. The process, which exploits the differentialreactivity of silanes with glass and aluminum oxide, is schematicallyillustrated in FIG. 18. PEG-biotin silanization is specific to glassunder the conditions employed, thereby resulting in chemicalderivatization of only the ZMW bottom surface. The aluminum layer isthen passivated using a polyelectrolyte multilayer, in this example, a2.5× multilayer of PAA/PEI/PAA/PEI/PAA (where PAA is poly(acrylic acid)and PEI is poly(ethyleneimine)). Biotin tagged polymerase is rejected bythe polyelectrolyte multilayer but binds to the biotinylated PEG surfacevia avidin chemistry, thereby resulting in biased immobilization of thepolymerase to the bottom surface of the ZMW. In addition, thepolyelectrolyte multilayer limits nonspecific binding of nucleotideanalogs to the aluminum layer.

Biased immobilization of polymerase on the bottom surface of ZMWs wasaccomplished as follows. ZMW chips are cleaned in an oxygen plasma for 2minutes at 2 torr (medium power setting). The PEG-Biotin silanization iscarried out for 3 hours at 4° C. using a mixture of PEG methoxy silaneand Biotin-PEG silane (Polymer Source Inc.) in 270:1 (w/w)ethanol:methanol solvent. The samples are rinsed with methanol,sonicated for 3 minutes in hot (70° C.) water, and washed with coldwater. The polyelectrolyte procedure consists of consecutive immersionof the chips for 5 minutes at room temperature in 20 mg/ml Polyacrylicacid and Polyethylenimine (Sigma-Aldrich, pH 7.5 adjusted with HCl),each step followed by 3× rinsing with water, in the order:PAA/PEI/PAA/PEI/PAA. The last wash is with 5 volume equivalents ofwater.

Nonspecific binding of four nucleotide analogs to the biasedimmobilization surface (ZMW chip treated with the mixture of PEG-silanesfollowed by polyelectrolyte multilayer formation) and to a controlsurface (a plasma-PDMS treated chip) was compared (FIG. 19). Theplasma-PDMS treatment used on the control chip removes bias because itcoats the entire structure with a uniform layer; see InternationalApplication Number PCT/US 2006/045,429 filed Nov. 27, 2006. Chips wereincubated with a mixture of fluorescently labeled nucleotide analogs(A488-dA4P, FAM-A532-dG4P, FAM-A594-dT4P, A633-dC4P, 5 μM each; see,e.g., U.S. patent application Ser. No. 11/645,223 for analognomenclature), and subjected to laser illumination. Movies were acquiredfor 1 minute at 100 fps camera speed. Fluorescence traces were analyzedby a custom-build analysis software, using a threshold algorithm todetermine the number of non-specific adsorption events shown in thegraph for each, spectrally separated analog. As shown in FIG. 19, thebiased immobilization surface is as good at preventing nonspecificanalog binding as is the plasma-PDMS surface (which exhibits goodnonspecific binding characteristics). Analog binding to an untreatedsurface was not quantified, since the analogs bind the untreated surfaceto such an extent that single pulses cannot be identified.

Polyelectrolyte multilayer deposition on an aluminum surface inhibitsnonspecific binding of polymerase, as illustrated in FIGS. 20A-20B.Essentially no DNA synthesis is observed on the aluminum surface treatedwith a 2.5× PAA/PEI/PAA/PEI/PAA polyelectrolyte multilayer (FIG. 20A),while DNA is produced over the entire surface of a control surface nottreated with the polyelectrolyte multilayer (FIG. 20B). Polymerizationreactions were carried out as follows: 100 nM polymerase was bound toNeutravidin (present in excess at 150 nM) in a BF-300 buffer containing25 mM Tris-acetate, pH 7.5, 300 mM potassium acetate, 0.05% Tween 20 and5 mM dithiothreitol for 30 minutes at 4° C. The solution was diluted toan effective potassium acetate concentration of 150 mM by 2-folddilution with the same buffer as above but lacking potassium acetate(BF-0). The polymerase/Neutravidin mixture was incubated for 30 minutesat 4° C. on the ZMW chip, and washed 3× with BF-150 buffer (the samebuffer as BF-300 but including 150 mM potassium acetate). Template at100 nM was added for 20 minutes at 4° C., in reaction buffer (50 mM Trisacetate, pH 7.5, 75 mM potassium acetate, 20 mM ammonium sulfate, 0.05%Tween 20 and 5 mM dithiothreitol) supplemented with 4 mM EDTA. Templatesolution was removed and the extension reaction mixture was added,containing 0.7 mM MnCl₂, 10 μM of each dATP, Alexa Fluor ChromaTide488-dCTP (Invitrogen), dGTP and dTTP in reaction buffer. DNA synthesisproceeded for 10 minutes at room temperature, followed by 5× washingwith BF-150 supplemented with 1 mM EDTA. ChromaTide nucleotideincorporation into DNA was visualized on a wide-field fluorescencemicroscope (Olympus), using a 60×0.9 NA physiology objective lens toimage the top (solution) side of the ZMW chips, and a 60×1.2 NAobjective lens for the bottom side.

The biased immobilization procedure (treatment of the ZMW chip with themixture of PEG-silanes followed by polyelectrolyte multilayer formation)results in selective immobilization of the polymerase within thewaveguides. Polymerization reactions were carried out as described inthe preceding paragraph on a biased immobilization ZMW chip and on acontrol ZMW chip (uniformly coated, with a plasma-PDMS layer underneathfollowed by PEG-methoxy/Biotin-PEG silane derivatization). Images of thebiased immobilization ZMW array, as well as images of the control array,are shown in FIG. 21. As shown in the control (Column II), bottom sideillumination (Row B) shows the presence of a significant amount of DNAwithin the waveguide structures, while top side illumination (Row A)shows a uniform layer of DNA produced over the entire surface of thearray. For the biased immobilization ZMW array (Column I), in contrast,both the bottom side (Row B) and top side (Row A) show a similar patternof DNA presence within specific waveguides. Further, as can be seen,there is little DNA present upon the upper surface other than withinwaveguides in the array, showing a substantial reduction from the highlevel of DNA synthesis present in the control experiment. Also of noteis that the waveguides showing DNA presence from the upper surface trackto the same waveguides showing DNA presence from the lower surface,indicating that DNA synthesis is occurring within the waveguidestructure, and not outside the waveguide core; see FIG. 22, in which animage of the top surface of a biased immobilization ZMW array (Panel I)and an image at the bottom surface of the same array (Panel II) areoverlaid (Panel III).

These results indicate that the polyelectrolyte multilayer is relativelynon-sticky to nucleotide analogs and that the polyelectrolyte multilayerpassivates well against polymerase binding to aluminum surfaces.Differential PEG-biotin-silane chemistry, followed by polyelectrolytemultilayer passivation, yields biased immobilization of the polymerasewith high contrast.

Example 4: Selective Immobilization and Passivation Using a PhosphonicAcid

Deposition of polyvinylphosphonic acid (PVPA) onto untreated ZMWsresults in a ZMW that is passivated from nonspecific protein (e.g.,neutravidin and polymerase) and nucleotide analog binding to thealuminum surface. PVPA is specific to aluminum and does not affect theSiO₂ bottom surface of the ZMW, which can be used for nonspecificcapture agent or polymerization immobilization or subsequentderivatization (e.g., by silanization or binding of compounds such asPLL-PEG) for specific polymerase deposition.

Treatment of a mixed material substrate (e.g., 100 nm aluminum film onglass) with PVPA results in immobilization of a neutravidin captureagent preferentially on the SiO₂, rather than the aluminum, portion ofthe substrate, as illustrated in FIG. 23. On a substrate not treatedwith PVPA, more neutravidin is deposited on the aluminum portion of thesubstrate (Panel I Row A) than on the SiO₂ portion (Panel I Row B). On aPVPA-treated substrate, in contrast, neutravidin is immobilizedpreferentially on the SiO₂ portion of the substrate (Panel II Row B),while little neutravidin sticks to the aluminum portion of the substrate(Panel II Row A).

To assess neutravidin binding, chips are cleaned from a protectivephotoresist layer by first rinsing them in acetone, followed by rinsingin isopropanol and drying with a stream of nitrogen. They are cleaned ina plasma cleaner (Harrick) for 2 minutes at 2 torr (medium powersetting). PVPA treatment proceeds on a heat block set to 90° C., thechips are put on the heat block, and 90° C. PVPA solution (molecularweight 24,000, from Polysciences Inc. (Warrington, Pennsylvania), 25%stock diluted to 2% working solution concentration in water) is put onthe chip for 2 minutes, followed by rinsing with water. Excess water isblown away by a stream of nitrogen, followed by heat treatment for 10minutes at 80° C. in a dry oven. 40 nm A488-Neutravidin latex beads(Invitrogen) are diluted to 0.01% in buffer (50 mM MOPS-acetate, pH 7.5,75 mM potassium acetate, 5 mM DTT) and incubated with the chips for 15minutes at room temperature. The chips are rinsed with water and imagedon a wide-field fluorescence microscope, using a 60×0.9 NA physiologyobjective lens (Olympus).

PVPA treatment reduces nucleotide analog binding, as illustrated in FIG.24. ZMW chips were treated with PVPA, and nonspecific binding ofnucleotide analogs to the chips was analyzed as described above inExample 3. As shown in FIG. 24, the analogs exhibit considerablenonspecific binding to an untreated ZMW (Panel II), while little analogbinding to a PVPA-treated ZMW is observed (Panel I).

Although described in some detail for purposes of illustration, it willbe readily appreciated that a number of variations known or appreciatedby those of skill in the art may be practiced within the scope ofpresent invention. Unless otherwise clear from the context or expresslystated, any concentration values provided herein are generally given interms of admixture values or percentages without regard to anyconversion that occurs upon or following addition of the particularcomponent of the mixture. To the extent not already expresslyincorporated herein, all published references and patent documentsreferred to in this disclosure are incorporated herein by reference intheir entirety for all purposes.

1-13. (canceled)
 14. A substrate comprising: a plurality of nanoscalewells disposed through an opaque layer, each well having a bottomsurface formed from a transparent layer underlying the opaque layer;wherein the wells comprise a coupling group bound to the bottom surfaceand located in an observation region; and wherein density of thecoupling group on the bottom surface of the wells is 100 times or moregreater than density of the coupling group on the opaque layer'ssurface.
 15. The substrate of claim 14, wherein the transparent layercomprises SiO₂.
 16. The substrate of claim 14, wherein the opaque layercomprises a metal or metal oxide.
 17. The substrate of claim 14, whereinthe opaque layer comprises aluminum.
 18. The substrate of claim 17,wherein the transparent layer comprises SiO₂.
 19. The substrate of claim14, wherein the opaque layer is passivated with a compound comprisingone or more phosphonic acid groups.
 20. The substrate of claim 14,wherein density of the coupling group on the bottom surface of the wellsis 1000 times or more greater than density of the coupling group on theopaque layer's surface.
 21. The substrate of claim 14, wherein thecoupling group comprises one or more functional chemical moietiesselected from the group consisting of an amine group, a carboxyl group,a hydroxyl group, and a sulfhydryl group.
 22. The substrate of claim 14,wherein the coupling group comprises biotin.
 23. The substrate of claim14, wherein the coupling group comprises avidin, streptavidin, anantibody, an antibody fragment, or a lectin.
 24. The substrate of claim14, wherein the coupling group comprises a nucleic acid.
 25. Thesubstrate of claim 14, wherein a molecule of interest is coupled to thecoupling group.
 26. The substrate of claim 25, wherein the molecule ofinterest is a macromolecule.
 27. The substrate of claim 25, wherein themolecule of interest is an enzyme.
 28. The substrate of claim 27,wherein the enzyme is a nucleic acid polymerase.
 29. The substrate ofclaim 14, wherein the wells comprise a solution comprising one or morenucleotides or nucleotide analogs.
 30. The substrate of claim 14,wherein the wells are zero mode waveguides.
 31. A system comprising: thesubstrate of claim 14; an illumination source that provides illuminationto the observation regions through the transparent layer; and an opticaldetector that receives electromagnetic radiation emitted through thetransparent layer from the observation regions.
 32. A method ofanalyzing a molecule of interest that is selectively immobilized on asubstrate, comprising: a) providing the substrate of claim 14 andcoupling the molecule of interest to the coupling group, therebyselectively immobilizing the molecule of interest on the substrate; andb) after selectively immobilizing the molecule of interest on thesubstrate, providing illumination to and receiving electromagneticradiation from the observation region of one or more of the nanoscalewells through the transparent layer.
 33. The method of claim 32, whereinthe transparent layer comprises SiO₂ and the opaque layer comprisesaluminum.